Biochimica et Biophysica Acta 1822 (2012) 690–713

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Review Oxidative stress and antioxidant therapy in cystic fibrosis☆,☆☆

Francesco Galli a,⁎, Andrea Battistoni b, Roberto Gambari c, Alfonso Pompella d, Alessandra Bragonzi e, Francesca Pilolli a, Luigi Iuliano f, Marta Piroddi a, Maria Cristina Dechecchi g, Giulio Cabrini g a Department of Internal Medicine, Laboratory of Clinical Biochemistry and Nutrition, University of Perugia, Italy b Department of Biology, University of Rome Tor Vergata, Italy c Department of Biochemistry and Molecular Biology, University of Ferrara, Italy d Dept. of Experimental Pathology & BMIE University of Pisa Medical School, Pisa, Italy e Infections and cystic fibrosis unit, San Raffaele Scientific Institute, Milan, Italy f Department of Medico-Surgical Sciences and Biotechnology, Vascular Biology & Mass Spectrometry Lab, Sapienza University of Rome, Italy g Laboratory of Molecular Pathology, Department of Pathology and Diagnostics, University Hospital of Verona, Italy article info abstract

Article history: Cystic fibrosis is a lethal autosomal recessive condition caused by a defect of the transmembrane conductance Received 20 October 2011 regulator gene that has a key role in cell homeostasis. A dysfunctional cystic fibrosis transmembrane conduc- Received in revised form 16 December 2011 tance regulator impairs the efflux of cell anions such as chloride and bicarbonate, and also that of other solutes Accepted 17 December 2011 such as reduced glutathione. This defect produces an increased viscosity of secretions together with other met- Available online 28 December 2011 abolic defects of epithelia that ultimately promote the obstruction and fibrosis of organs. Recurrent pulmonary infections and respiratory dysfunction are main clinical consequences of these pathogenetic events, followed by Keywords: fi Cystic fibrosis pancreatic and liver insuf ciency, diabetes, protein-energy malnutrition, etc. This complex comorbidity is asso- Antioxidant ciated with the extensive injury of different biomolecular targets by reactive oxygen species, which is the bio- Oxidative stress chemical hallmark of oxidative stress. These biological lesions are particularly pronounced in the lung, in which Reactive oxygen species the extent of oxidative markers parallels that of inflammatory markers between chronic events and acute exac- Inflammation erbations along the progression of the disease. Herein, an abnormal flux of reactive oxygen species is present by Glutathione the sustained activation of neutrophils and other cystic fibrosis-derived defects in the homeostatic processes of pulmonary epithelia and lining fluids. A sub-optimal antioxidant protection is believed to represent a main con- tributor to oxidative stress and to the poor control of immuno-inflammatory pathways in these patients. Ob- served defects include an impaired reduced glutathione metabolism and lowered intake and absorption of fat-soluble antioxidants (vitamin E, carotenoids, coenzyme Q-10, some polyunsaturated fatty acids, etc.) and oligoelements (such as Se, Cu and Zn) that are involved in reactive oxygen species detoxification by means of enzymatic defenses. Oral supplements and aerosolized formulations of thiols have been used in the antioxidant therapy of this inherited disease with the main aim of reducing the extent of oxidative lesions and the rate of lung deterioration. Despite positive effects on laboratory end points, poor evidence was obtained on the side of clinical outcome so far. These aspects examined in this critical review of the literature clearly suggest that fur- ther and more rigorous trials are needed together with new generations of pharmacological tools to a more ef- fective antioxidant and anti-inflammatory therapy of cystic fibrosis patients. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease. © 2012 Elsevier B.V. All rights reserved.

1. Introduction in the apical membrane of epithelial cells that line mucous mem- branes and submucosal glands [1]. Several mutations have been Cystic fibrosis (CF) is a lethal autosomal recessive disorder caused identified to cause this gene defect with the Phe508del, or ΔF508, by a single gene defect. This was identified in 1989 to map on the as one of the most common mutations in Caucasians. The prevalence chromosome 7 and to correspond to the gene coding for the trans- at birth varies in the different regions according with ethnic back- membrane conductance regulator (CFTR) that is mainly expressed ground, from roughly 1 in 3000 white Americans and northern

☆ This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease. ☆☆ For the “Working Group on Inflammation in Cystic Fibrosis” of the Italian Society for Cystic Fibrosis and the Italian Cystic Fibrosis Research Foundation. ⁎ Corresponding author. E-mail addresses: [email protected] (F. Galli), [email protected] (A. Battistoni), [email protected] (R. Gambari), [email protected] (A. Pompella), [email protected] (A. Bragonzi), [email protected] (F. Pilolli), [email protected] (L. Iuliano), [email protected] (M. Piroddi), [email protected] (M.C. Dechecchi), [email protected] (G. Cabrini).

0925-4439/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2011.12.012 F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 691

Europeans to 1 in 350,000 in Japan. Mutations are grouped in 6 clas- conspire with further mechanisms to worsen the prognosis of this ses based on the type of defect caused on CFTR protein metabolism inherited disorder (recently reviewed in [10]). and function. Several physiological processes affected by these muta- In view of these aspects, the CF patient is assumed to have a higher tions are related to the role of CFTR as anion channel. This mainly antioxidant demand. This has provided the rationale for the systematic regulates chloride efflux, but other and larger anions such as reduced investigation of antioxidant levels in blood and targeted tissues of CF glutathione cross the plasmalemma throughout this transmembrane patients, mainly the epithelium and lining fluids of the airways, and protein widely expressed in diverse epithelial tissues. Other ion to plan for antioxidant interventions that might rescue specificdefects transport systems are under its influence, such as bicarbonate of these patients. These also include the use of anti-inflammatory anion and sodium channels, so that a defective CFTR can impair agents and nutritional formulations which can produce an “antioxi- several processes such as cell volume and pH regulation, transepithe- dant effect”, i.e. the lowering of oxidative stress indices, as a result of lial transport, membrane conductance, and the GSH-related antioxi- their direct or indirect action. Despite a number of promising in vitro dant and detoxification activity in the extracellular milieu [2,3]. and pre-clinical observations, antioxidants used as oral supplements CFTR dysfunction is associated with an altered fluid and electrolyte or directly administered in the CF airways have failed to provide con- composition of secretions, their increased viscosity and progressive vincing evidence at the clinical level. Future efforts are required to iden- obstruction and fibrosis of organs [4]. The severity of these CF symp- tify more advanced agents and therapeutic strategies that may enhance toms varies independently of the type and number of mutations secondary prevention and chemotherapy of airway inflammation and diagnosed, suggesting that CFTR gene and its mutations interact oxidative stress in CF patients. Advances in the approaches capable of with other genes at the transcriptional and post-translational level improving nutritional status and antimicrobial therapy are of main to influence a wide series of physiological processes. Lung, pancreas relevance to further ameliorate quality of life and survival rates in CF. and liver are severely affected by these events, and recurrent infec- These aspects will be discussed in this manuscript with the aim of tions of the airways together with pancreatic insufficiency and diabe- providing an updated review of the literature as well as of strategies tes are most common conditions secondary to CF [1]. and future directions of antioxidant therapies in CF patients. The presence of a defective CFTR appears to produce a redox im- balance in epithelial cells and extracellular fluids and to cause an ab- 2. Inflammatory pathways and oxidative stress in CF normal generation of reactive oxygen species (ROS). A constitutive defect of GSH metabolism together with a lowered intake and absorp- 2.1. Progressive inflammatory damage in CF lungs and the contribution tion of fat-soluble antioxidant vitamins (vitamin E and carotenoids) of oxidants could contribute to a defective antioxidant protection, which is be- lieved to exacerbate oxidative stress indices along with the progres- Evidence supporting the occurrence of oxidative stress in CF is by sion of clinical status [5–7]. The development of inflammatory and now established and extensive [6,10–12]. As introduced above, CF- degenerative lesions in target tissues such as lung, pancreas and related defects of the pulmonary epithelium and a sustained PMN liver further exacerbate the shift from normal to abnormal flux of activation by recurrent infections create the conditions for an abnor- ROS in several organs, thereby leading to develop systemic oxidative mal flux of reactive oxygen species (ROS) in the CF lung (Fig. 1) be- stress. This is a chronic-degenerative trait common to other and tween events of acute and chronic inflammation. Abnormalities of severe inflammatory diseases such as chronic kidney disease and markers of ROS activity and inflammation are most evident during some auto-immune syndromes (reviewed in [8,9]), which may acute respiratory exacerbations and show improvement with the

Fig. 1. Oxidative unbalance in conductive airways of patients affected by cystic fibrosis. airway surface liquid (ASL) in CF bronchi is characterized by 1. increased concentration of reactive oxygen species (ROS), 2. lowered levels of glutathione (GSH) and 3. reduced nitric oxide (NO). The net increase of pro-oxidative species in ASL, as a result of derangements of both neutrophils and bronchial epithelial cells, contributes to the progressive lung tissue damage and to the amplification of the inflammatory response in CF airways. This is characterized by the release of chemokines and cytokines (e.g. IL-8 and IL-6, respectively). NOX, NADPH oxidase; MPO, mieloperoxidase; DUOX, dual oxidase; LPO, lactoperoxidase, IL, interleukin. 692 F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 intensive treatment of the infection [13–15]. The fact that relapses or is continuous and even independent of the presence of overt bacterial successful therapy of infection do not normalize these markers dem- infection [33]. Interestingly, it was proposed that the efficiency of the onstrates the presence of a chronic inflammatory syndrome that is DUOX/LPO defense system is dependent on the ion transport function intrinsic to the CF defect. In this context, ROS may lose their physio- of the CFTR Cl− channel, which could also intervene both in the con- − − logical role in the killing of pathogens, to turn into toxic effectors re- ductive secretion of SCN (for LPO function) and of HCO3 (for pH sponsible for the damage of the pulmonary epithelium as well as of adjustment) into the ASL [34]. However, more recent ex vivo observa- other components of the lung parenchyma and lining fluids. Impor- tions, while confirming a positive role of DUOX/LPO system in produc- tantly, ROS can modify the thiol homeostasis of extracellular fluids ing OSCN− as a general defense mechanism of the airways, do not and epithelia [16] and promote the activation of MAPK signaling directly relate SCN− concentrations in ASL with CFTR function [35]. − pathways [17], which regulate both the NFkβ-dependent and The LPO-mediated scavenging of H2O2 has suggested a role of SCN -independent transcription of pro-inflammatory genes and other mo- as physiological antioxidant of ASL [36], which may be defective in − lecular effects associated with the immuno-inflammatory imbalance CF. Besides this role of LPO and SCN , an abnormal flux of H2O2 in observed in the CF lung. the CF airways may also depend on other factors that are associated Hallmark of the chronic inflammatory lung disease in CF is the with an altered metabolism of ROS. For instance, lowered levels of Lac- release of chemokines, mainly interleukin (IL)-8 [18,19], leading to toferrin (LF) have been described in CF secretions [37]. This iron- the neutrophil recruitment in the bronchial lumen (see [20] for a chelating homologue of transferrin contained in the granules of neu- review). Whether CF lung inflammation arises independently and trophils is also secreted by several mucosal tissues in biological fluids before bacterial infection remains to be fully established, although to contribute antimicrobial effects by a variety of mechanisms includ- IL-8 and pro-inflammatory cytokines have been found in bronchoal- ing the chelating activity of iron as Fenton chemistry catalyst [38]. veolar lavage fluids of CF infants even before the onset of an overt As regards the progression of CF lung disease, in the early phases, bacterial infection [21]. Although directed against infective agents, before the onset of chronic bacterial colonization, epithelial DUOX the chronic inflammation in CF lungs is largely recognized as mainly continuously releases H2O2, thus being a predominant source in responsible for the progressive tissue damage leading to respiratory respect to the NADPH oxidase from phagocytes. The latter is mainly insufficiency. Dissection of the pathophysiology of CF chronic lung active when the respiratory burst is “triggered on demand” by infec- inflammation should take into account the bronchial epithelial cells tious components. On the other side, in advanced phases of chronic expressing the mutated CFTR protein, the polymorphonuclear neu- infection of CF lungs, neutrophil-derived ROS are predominant [32], trophils recruited into the bronchial lumens and the bacterial infec- due to neutrophil activation as well as to the decreasing number of tion itself, with special regards to Pseudomonas aeruginosa, the most H2O2-producing ciliated cells, which are reduced by extensive apo- common gram negative microorganism which colonizes CF airways ptosis and tissue remodeling. A further reduction of epithelial DUOX [22]. Thus, novel anti-inflammatory therapies against the progressive activity has been observed as a result of infection with P. aeruginosa damage of the CF respiratory tissue should be mainly aimed i) to re- in conductive airways. P. aeruginosa-derived toxin pyocyanin in fact duce the excessive recruitment of neutrophils, by intervening on the inhibits DUOX-dependent H2O2 production by consuming intracellu- transmembrane signaling pivoting the excessive expression of IL- lar NADPH, which represents an interesting adaptive mechanism to 8 [23–26], ii) to inactivate proteases released by the neutrophils con- downregulate innate anti-bacterial defenses [39]. tinuously activated by bacterial products [27] and iii) to circumvent the effect of the unbalanced production of oxidants, deriving from 2.3. Redox disturbances of CF airways: the role of GSH, NO and H2O2 both phagocytes and bronchial epithelial cells [22,28–30]. Identifica- tion of oxidants produced in the CF airway tract is of high importance, Intracellular impairment of redox balance between oxidants and in order to identify novel molecular targets for specific pharmacolog- anti-oxidants has been proposed to occur in CF bronchial epithelial ical intervention. cells, although a significant difference in respect to normal CFTR- expressing cells is still controversial and debated [40]. Three major 2.2. Respiratory epithelial cells and neutrophils as sources of oxidants in issues have been investigated concerning the intracellular redox bal- the CF lung ance in CF bronchial cells, namely i) a defect in GSH homeostasis [41,42], ii) an alteration of nitric oxide (NO) metabolism and iii) an

It is largely accepted that neutrophils migrating inside the CF imbalance of intracellular H2O2 production. bronchial lumina release large amounts of reactive oxygen species As far as GSH is concerned, the defective CFTR channel function − (ROS), including the superoxide anion (O2 •), hydrogen peroxide has been proposed to lead to a lowered cell content of this tripeptide, (H2O2) and the hydroxyl free radical (OH), mainly by the activation which is crucial to control the flux of H2O2 in both the CF bronchial of the NADPH oxidase (NOX). To such exaggerated ROS production epithelial cells and lining fluids. This aspect – discussed in detail contribute both the continuous interaction of neutrophils with bacte- below in this review – is associated with characteristic defects of ria and bacterial degradation products and the inability to engulf bac- GSH-related enzymes and may represent a key underlying factor in teria in biofilms, leading to a condition of “frustrated phagocytosis”. the oxidative stress of CF airways. Neutrophils are therefore recognized as a major source of ROS in Altered NO concentration has been found in chronic respiratory the airway surface liquid (ASL) of young children with CF [14,31]. diseases such as bronchial asthma and chronic obstructive pulmonary However, bronchial ciliated and alveolar type II epithelial cells by disease [43], and reduced NO concentrations have been observed in themselves are able to produce significant amounts of ROS, through the bronchial airways of patients affected by CF, which directly corre- the two isoforms of NADPH oxidase expressed in the apical membrane lated with worsening of lung function [44]. Thus NO concentration in of these epithelial cells, namely DUOX1 and DUOX2 [32]. A major pro- CF lung exhalate has been subsequently tested as a possible marker of posed function of DUOXs is to support lactoperoxidase (LPO), which is pulmonary exacerbations and/or the inflammatory/infective status in turn released by goblet and submucosal gland airway cells, to gen- and its fluctuations over time [45,46]. erate bactericidal hypothiocyanite (OSCN−) starting from thiocyanate This opened the way to further analysis of the mechanisms of this − (SCN ) and H2O2. Thus the DUOX/LPO coupled system of the respira- derangement, and it has been proposed that an excessive production tory epithelial cells parallels the NOX/mieloperoxidase (MPO) system of asymmetric dimethylarginine, an inhibitor of endogenous NO of phagocytes in releasing and processing oxidants in the ASL. While Synthase (NOS), could be involved in the reduced concentration the NOX/MPO system of phagocytes is mainly activated in the of NO in CF airways [47–49]. Possible corrective therapies, such as infection-induced respiratory burst, the release of ROS from epithelia the inhalation of L-arginine, have provided preliminary evidence of F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 693 correction of the defective NO concentration and improvement of Moreover, oxidants could be synergic in the induction of mucins, as lung function in CF patients [49–51]. promoted by neutrophil elastase, which further impairs ASL fluidity Although exhaled NO is decreased in CF, increased immunohisto- in CF [60]. Finally, bacterial infection with P. aeruginosa strains releas- chemical staining for nitrotyrosine was demonstrated in lung tissues ing the toxin pyocyanin (PCN) has been shown to reduce ion trans- from CF patients [52]. Therefore, a decreased production or accelerat- port through the CFTR channel, thus potentially counteracting the ed metabolism of NO could be present in association with an abnor- therapeutic effects of correctors and potentiators of mutated CFTR mal reactivity of this radical and its derived species (NOx) toward protein [61,62]. In summary, excessive oxidants in CF conductive air- biomolecular components of the CF airways. Peroxynitrite is one of ways have different negative effects in the amplification of the al- the most relevant mediators of the biological activity of NO with ready excessive lung inflammation and secretion of mucin, together toxic properties and damaging activity on several biomolecules [53]. with a direct deleterious effect on CFTR channel function. A third emerging issue takes into consideration the expression of different enzymatic systems affecting the redox balance in CF bron- 2.5. Oxidative stress and surfactant chial epithelial cells. For instance, intracellular concentration of H2O2 has been found abnormally elevated in both immortalized bronchial Oxidative stress and inflammation in cystic fibrosis can affect sur- and primary nasal epithelial cells derived from CF patients, both factant biophysical activity thus leading to early alterations of lung in the presence and the absence of proinflammatory cytokines [54]. function in patients with CF [63]. Altered phospholipid-to-protein ra- This has been accompanied by a marked decrease of expression tios and phospholipid subclasses, a modified fatty acid profiles, and of proteins regulating H2O2 levels, such as thioredoxin 1 (TRX-1), decreased association of proteins such as SP-A with lipid components glutathione-S-transferase pi (GST-pi), peroxyredoxin (PRDX) 6, of isolated surfactant, indicate that components of this fluid are con- TRX-dependent peroxide reductase (PRDX-1), catalase and, con- siderably altered and dysfunctional in lower respiratory tract secre- versely, a significant increase of Mn superoxide dismutase (SOD2) tions of CF patients [64]. [54]. Interestingly, to link these modifications with CF specific charac- Oxidative damage of surfactant may involve both lipid and protein teristics, dysfunctional CFTR channel was found associated with components. Alteration of lipid components can in turn generate reduced activity of the transcription factor Nrf-2 (nuclear factor- toxic lipid species with cytotoxic activity towards nearby epithelial erythroid 2 p45 subunit-related factor 2), which could at least in cells [65]. Altered protein components have been shown in cystic part explain the differential expression of the enzymatic systems fibrosis [66]. Notably, surfactant protein D, which is an important in- resulting in the elevated intracellular steady-state concentration of nate host defense molecule, becomes unable to agglutinate bacteria

H2O2 found in CF nasal and bronchial epithelial cells [54]. In synthesis, when it is modified by oxidation, which facilitates pathogen coloniza- the ASL of CF patients during advanced stages of the lung disease con- tion in the lung [67]. In a cross-sectional analysis of CF patients with tains elevated concentrations of ROS, mainly derived from neutro- mild lung disease, reduced surfactant activity was correlated to in- phils migrated into the airway lumen, and a reduced concentration creased neutrophilic airway inflammation, but not to lung function of NO, which can strongly contribute to respiratory tissue injury [68]. So far, longitudinal measurements of surfactant function in CF together with the proteases released by the activated neutrophils. patients are lacking and it remains unclear how these alterations The homeostatic role of the GSH-related defenses appears constitu- relate to progression of airway inflammation as well as to the rate tively impaired by the dysfunctional CFTR, thus increasing suscepti- of decline of pulmonary function [69]. bility to develop oxidative stress and lung tissue degeneration (Fig. 1), as described in detail in the sections below. 2.6. Laboratory indices of oxidative stress in CF

2.4. Conductive airway epithelium as target of ROS Appropriate biochemical and clinical tools are of importance for the monitoring of antioxidant therapies in CF, and a crucial aspect is Oxidants can target different biomolecules to damage epithelial the selection of proper biomarkers and protocols to assess biological cells and extracellular fluids of the airways. Lipid peroxidation pathways of oxidation. and post-translational modifications of proteins on both cell mem- Pancreatic insufficiency and a diminished bile acid pool cause mal- branes and extracellular targets are common biomarkers of this absorption of important essential nutrients and other dietary compo- damage, which can occur by the direct reaction between ROS and bio- nents in CF. Of particular significance is the malabsorption of fat- molecules or through the formation of second-generation reactive soluble antioxidants such as carotenoids, tocopherols and coenzyme byproducts [9,55]. All levels of this interaction between ROS and bio- Q-10 (CoQ-10), which act as chain breakers in the peroxidation reac- logical components can produce toxic and bioactive intermediates. tions of polyunsaturated lipids. Accordingly, lipid peroxidation is one Oxidants are known to activate second messengers through phospho- of the main signs encountered in the CF plasma, buccal mucosal cells, lipases A2, C and D, and to induce the production of cytokines and breath condensate and BALF as measured by the non-enzymatic mucins, a series of molecular events that contribute to progressive oxidation product of arachidonic acid 8-iso prostaglandin F2α obstructive disease and reduction of lung function [56]. Besides the [5,70–73]. Elevations of this and other eicosanoids in human body direct oxidative damage to cellular structures of the bronchial epithe- fluids and tissues have been found in a diverse array of human disor- lial cells, the excessive concentration of oxidants in CF, both in the ASL ders, including atherosclerosis, diabetes, obesity, cigarette smoking, lining the apical membranes and inside the bronchial cells, has been neurodegenerative diseases, and many others [74]. Further, treat- studied in respect to the regulation of the inflammatory response. ments for some of these conditions, including antioxidant supple- ROS are often considered a sort of second messengers in activating mentation, have been shown to decrease the levels of this class of the Nuclear Factor (NF)-κB, which is in turn involved in the activation biomarkers. In CF patients, respiratory exacerbations increase plasma of transcription of several proinflammatory cytokines and chemo- levels of 8-iso-F2α [70], the levels of which in the breath condensate kines [57]. For instance, it has been shown that H2O2 partly controls negatively correlate with respiratory function data [71]. At the same NF-κB activation by IL-1beta, by facilitating the activation of NIK time, successful in vivo antioxidant therapy by GSH inhalation has and subsequent phosphorylation of IKKβ [58]. In this respect, a direct been shown to decreased PGE levels in BALF, in association with link has been proposed between the excessive production of intracel- changes in the number and activity of leukocyte subpopulations re- lular H2O2 and the elevated expression of IL-6 and IL-8, the most sponsible for lung inflammation [73]. abundant pro-inflammatory cytokine and neutrophilic chemokine Oxysterols, a biomarker of cholesterol oxidation, were found to found in CF airways [54], which has been further confirmed [59]. increase in CF plasma as a further proof of the abnormal lipid 694 F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 metabolism and increased susceptibility to oxidation of lipoprotein the airway antioxidant defense. Indeed, these proteins are potent in lipids in CF patients [7]. Importantly, an increased oxidative burden vitro ROS scavenges [83] and their synthesis is upregulated upon ox- of lung and blood lipids may produce bioactive lipid products that idative stress via epidermal growth factor receptor (EGFR) transacti- further sustain CF symptoms. Besides to 8-iso-F2α and other ana- vation [84]. logues with some bioactivity [74], arachidonic acid oxidation may GSH, a major component of cellular antioxidant defenses, exerts im- contribute to persistent platelet activation and pulmonary dysfunc- portant functions related to its electron-donating capacity, including tion in CF via generation of bioactive isoeicosanoids [75], which pro- protection from the damaging effects of ROS and regulation of a pletho- vides sufficient rationale for a prevention therapy with fat soluble ra of cellular events, such as gene expression, proliferation and differen- antioxidants such as vitamin E. Evidence has recently accumulated tiation, apoptosis and immune response [85]. GSH is synthesized by two on the systemic effects of oxysterols on various tissues and organs sequential ATP-dependent reactions catalyzed by γ-glutamylcysteine [76]. The role of this lipid oxidation product alone or in combination synthetase (recently, renamed glutamate-cysteine ligase) and GSH syn- with other factors could be further investigated in the context of thetase. The reaction catalyzed by γ-glutamylcysteine synthetase, i.e. mechanisms and clinical progression of multi-organ failure of CF the formation of γ-glutamylcysteine from glutamate and cysteine, is patients. the rate-limiting step in GSH synthesis and is feedback regulated by The impaired pancreatic and liver functions of CF patients repre- GSH itself. In fact, this enzyme represents an interesting case of redox- sent the underlying factor for a defective lipoprotein metabolism regulation of catalytic activity that is mediated by the reversible forma- and hypocholesterolemia, which exponentially increase the burden tion of disulfide bonds [86]. Oxidizing conditions causing GSH depletion of damage by cholesterol and other lipids due to reduced blood trans- promote the formation of a disulfide bond between the catalytic and the port of fat-soluble antioxidants with nascent VLDL particles. Besides regulatory subunits of the enzyme, leading to a conformational change oxysterol accumulation, plasma fatty acid composition is also affected which favors the binding of glutamate. In contrast, physiological levels [7], and an increased ratio between unsaturated and saturated fatty of GSH reduce this disulfide bond, thus explaining GSH feedback acid species may contribute to lower cellular antioxidant defenses. inhibition. Altogether, these findings suggest that lipid oxidation biomarkers Either in intracellular or extracellular compartments, GSH is pre- can provide a reliable measure of systemic and lung-specific oxidative dominantly found in the reduced form, although small amounts of stress in CF. the oxidized disulfide forms (GSSG or GSSR, where a GSH molecule Markers of protein damage are also detectable in the airways of is linked to a free or a protein thiol) can always be detected. GSSG is children with CF and their levels are observed to parallel the extent produced by the catalysis of glutathione peroxidase, during the of neutrophilic markers and lung dysfunction [14,31,66]. Bronchoal- detoxification from hydrogen peroxide and other peroxides, or by veolar lavage proteins undergo halogenation of Tyr residues, a the direct reactions of GSH with electrophilic compounds, such as radical-mediated process presumably depending on MPO enzyme ac- radical species. Despite the bulk of GSH synthesis occurring in the tivity and assessed through the analysis of 3-chlorotyrosine and 3- cytoplasm, GSH is distributed in intracellular organelles, including bromotyrosine. Thiocyanate and protein carbonyls are also useful bio- the endoplasmic reticulum, mitochondria and nucleus. Under physio- markers to assess the inflammation-related injury of BALF proteins in logical conditions, the GSH to GSSG ratio in these compartments is CF [14,31]. high, frequently >100/1, but this may change under conditions of ox- idative stress [87]. A major exception is represented by the endoplas- 3. Antioxidants in CF mic reticulum, where GSSG is present at much higher levels to favor disulfide bond formation [88]. Interestingly, the compartmentaliza- 3.1. Glutathione and its related defense system tion of GSH in separate pools within organelles allows localized alter- ations in the balance between GSH and GSSG that may have 3.1.1. Defects in GSH homeostasis considerable functional and pathological significance [89]. This Other studies have pointed to alterations in the levels of extracel- could be particularly important in CF, in view of the above mentioned lular antioxidants in respiratory tract lining fluids [3]. In particular, studies showing that cultured CF cells have an apparent normal GSH/ the analyses of broncheoalveolar lavages (BAL) have revealed the GSSG content, but are characterized by a marked decrease in mito- presence of decreased levels of GSH in the alveolar epithelial lining chondrial GSH in association to elevated mitochondrial ROS [79,80]. fluid of CF patients. The normal level of extracellular GSH in the Different roles of high levels of GSH in the ELF may be hypothe- lung is 140 times that present in blood plasma, and the lung is a net sized, including: a) preventing inflammation and tissue damage by importer of circulating GSH [77]. As a result, GSH concentration in scavenging the ROS spontaneously generated in this highly oxidizing ELF is close to 400 μM, whereas significantly reduced GSH levels are environment, actively produced by neutrophils during inflammation present in adult CF patients [41]. Low levels of GSH have been ob- or originating from lipid peroxidation, b) regulating the redox status served in plasma and blood neutrophils [41,78] suggesting systemic of membrane proteins involved in the transduction of signals leading GSH dyshomeostasis in CF. Moreover, some studies have shown that to changes in the expression of genes involved in the immune re- at the cellular level, the CFTR mutation causes mitochondrial deple- sponse, c) controlling mucus viscosity by breaking disulphide bonds, tion of GSH [79,80]. The consequences of this defect are still difficult d) modulating the response to bacterial infections. This last possibili- to be understood due to our poor knowledge about the exact func- ty is suggested by the observation that GSH significantly increases tions of GSH in the lung, but there are reasons to believe that the in the ASL of wild type mice following P. aeruginosa infection, where- decrease of GSH in the ASL contributes to lung infection and inflam- as this response is not observed in CFTR mutant mice [90]. Interest- mation [42,81]. At the same time, it must be underlined that a recent ingly, some authors have suggested the ability of GSH to control P. study has shown that the GSH content in sputum samples is higher in aeruginosa growth and resistance to antibiotics, although these stud- CF patients than in healthy people, indicating that GSH deficiency in ies should be considered with some cautions due to the likely use of CF is restricted to the lower respiratory tract [82]. Several hypotheses unbuffered GSH [91]. have been proposed to explain such a local increase in GSH, including Although the decreased levels of GSH in ELF could be due to in- the possibility that it may derive from apoptotic neutrophils. The re- creased consumption during inflammation-related oxidative stress lease of high levels of GSH may be part of a compensatory mechanism [41,92], GSH deficiency in CF ELF likely derives from CFTR channel aimed at controlling disulfide bonds-mediated aggregation of mucins. dysfunction. In fact, comparable alterations in GSH extracellular con- Secretions of the upper respiratory tract contain abundant levels of tent characterize the lung of CFTR knockout mice [93], indicating that these glycosylated and cysteine-rich proteins, likely playing a role in this defect is correlated to mutations in CFTR. F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 695

In particular, ELF and lung tissue from CFTR knockout (Cftr KO, revealed that extracellular GSH inhibits the ability of Burkholderia B6.129P2-Cftrtm1Unc) and wild-type mice were compared for GSH cenocepacia to enter epithelial respiratory cells and reduces bacterial content and the activities of glutathione-related enzymes [93].In induced expression of proinflammatory cytokines (D'Orazio, Pacello the ELF, the concentration of GSH was significantly decreased in the and Battistoni 2011, unpublished results). Cftr KO mice compared to WT, whereas tissue concentrations of GSH were similar [92]. In the Cftr KO lung, the activities of glutathione reductase and glutathione peroxidase were increased, whereas the 3.2. GSH-based antioxidant therapies activity of γ-glutamyltransferase was unchanged. Two indicators of oxidative stress, thiobarbituric acid reactive substances (TBARS) and 3.2.1. Preclinical studies in animal models of CF 8-hydroxy-2-deoxyguanosine (8-OHdG), were also increased in the Mice genetically modified for the Cftr gene, along with acute and Cftr KO lung tissue [90]. These data support the hypothesis that a mu- chronic infection induced by CF-related pathogens, are a key asset in tation in the CFTR gene can affect the antioxidant defenses in the lung CF research. Although much has been learned through these CF and may contribute to the exaggerated inflammatory response ob- mouse models, limitations in the ability of this species to recapitulate served in CF. Thereby, CFTR could be considered as an important spontaneous lung disease and several other organ abnormalities actor of ELF antioxidant homeostasis and thus an intrinsic cause of seen in CF humans, including few airway mucous glands, have creat- oxidative imbalance in CF airways of human patients as well as Cftr ed a need for additional species on which to study CF [106].Tothis KO mice. end, pig and ferret CF models have been generated and are currently Moreover, CFTR belongs to the MRP/ABC family of proteins which being characterized [107,108]. These new larger animal models have includes several GSH transporters and some in vitro studies have in- phenotypes that appear to closely resemble human CF disease seen dicated that CFTR may mediate GSH export across membranes in newborns, and efforts to characterize their adult phenotypes are [2,3,94]. Despite these evidences, there is still debate about the phys- ongoing. However, mice have been the dominant species by which iological implication of CFTR in GSH transport outside the cells as to study CF disease processes in vivo and develop therapies for the other studies have raised the possibility that CFTR may not actually past two decades including GSH-based antioxidant treatment. conduct GSH, but regulate its transport indirectly through chloride Despite limitations and significant species differences between transport [85]. mice and humans, these models proved to be useful tools to mimic the initial and progressive bronchopulmonary infection typical of CF patients [109]. In particular, the model of chronic infection, which 3.1.2. In vitro studies suggesting a protective role of GSH in CF model challenge bacterial cells with agar as an immobilizing agent, has systems been extensively characterized, and induce the long-term persistence Possible protective roles of extracellular GSH in the CF lung have of the bacterial infection and lung pathology, including airway inflam- been long proposed and recent in vitro studies have provided further mation [110,111]. Lung pathology associated with chronic experimen- and more robust support to this clue. For example, it has been sug- tal infection resembled some aspects of the advanced chronic gested that GSH may control the levels of chlorinated compounds pulmonary disease at autopsy in CF patients [112–114]. Both naturally formed by the activity of myeloperoxidase, a neutrophil-released occurring and experimental infections frequently manifest broncho- protein abundantly present in CF patients secretions [95,96] and pre- pneumonia, bronchiectasis, mucus plugging, epithelial metaplasia, vent NK-κB activation [96]. Other studies have suggested that CFTR fibrosis, and alveolar exudates with inflammatory cells. Lymphoid mutant cells produce higher levels of proinflammatory cytokines in hyperplasia, which was prominent in the infected animals, was also response to P. aeruginosa diffusible material with respect to wild a common finding in the lungs of CF patients. In addition, significant type cells, through a mechanism involving the activation of NADPH differences with regard to weight loss, BAL neutrophil counts and oxidase. This effect may be significantly reversed by the addition of cytokine concentrations have been detected. Infected mice had a extracellular GSH [97]. rapid though transient rise in absolute neutrophil counts, TNF-α, IL- GSH could also play an important role in protecting the lung epi- 1β, IL-6, MIP-2, and KC in bronchoalveolar lavage (BAL) [110,115].In thelia from the toxic effects of pyocyanin (PCN), a redox-active exo- addition, the generation of CF mice has allowed the possibility of in toxin released by P. aeruginosa which is supposed to cause a variety vivo testing of novel therapies before entering in clinical trial. These of deleterious effects on the airway physiology [98]. PCN levels as include the pre-clinical evaluation of antibiotics and biotechnological high as 130 μM have been measured in pulmonary secretions of pa- drugs, as well as of natural and synthetic anti-inflammatory agents tients with CF and individuals with chronic bronchiectasis [99].At that reduce the excessive recruitment of neutrophils and the progres- concentrations within the range measured in the sputum from CF pa- sive damage of the respiratory tissue by the unbalanced production of tients, PCN induces a drastic reduction of intracellular GSH [100,101], oxidants. promotes death of cultured cells [100] and causes pathophysiological Regarding antioxidants, GSH and its pro-drug N-acetylcysteine alterations in the lung of wild type mice that are consistent with the (NAC) remain the so far most investigated antioxidant agents in CF changes observed in CF patients [102]. PCN toxicity likely derives and several strategies have been proposed to improve systemic and from its ability to accept electrons from cellular reductants and then lung GSH status of CF patients based on pre-clinical studies, including react with oxygen to generate superoxide and other ROS [103]. There- the administration of nebulized GSH by inhalation or the oral supple- fore, GSH deficiency is explained either by the reaction of GSH with mentation of GSH or NAC. Oral GSH administration can raise serum such oxidants or through the direct reaction of PCN with GSH that and lung tissue GSH levels in rodents [116,117]. In these studies leads to the formation of a PCN radical [103]. However, the electron GSH was administered to animals dissolved in saline or PBS. The transfer from GSH to PCN does not occur at neutral pH and recent ob- pharmacokinetic profile of an oral bolus dose of GSH (300 mg/kg) servations suggest that extracellular GSH provides significant protec- was determined in mice in other studies [118]. Plasma, ELF, BAL tion against the toxic effects of PCN [104]. Moreover, GSH can react cells, and lung tissue were analyzed for GSH content. There was a with PCN to form a stable adduct which is likely redox inactive rapid elevation in the GSH levels that peaked at 30 min in the plasma [105]. Although the relationships between extracellular GSH and and 60 min in the lung, ELF, and BAL cells after oral GSH dosing. Oral PCN are still contradictory, it is worth mentioning that extracellular GSH treatment produced a selective increase in the reduced and ac- GSH increases to millimolar levels in the ELF of wild type mice tive form of GSH in all lung compartments examined. Oral GSSG treat- infected with P. aeruginosa, indicating that GSH may be useful to re- ment (300 mg/kg) resulted in a smaller increase of GSH levels. To sist to bacterial colonization [90]. Interestingly, in vitro studies have evaluate the role of CFTR in this process, Cftr KO mice and gut- 696

Table 1 Intervention trials on antioxidant therapy in CF patients found at ClinicalTrials.gov databasea.

Rank Title PI Location Recruitment Interventions Age groups Phases; Study designsb Outcome measures Start and NCT ID number completion date enrolled

1Efficacy and safety study Griese Germany Completed Drug: C|A|S II 1. R|E • Differences between inhaled GSH Jul-07 00506688 of inhaled GSH in CF M. • reduced GSH sodium salt 138 2. Safety/eff and inhaled normal saline with May-10 patients • 0.9% saline (control) 3. PA respect to the area under the curve 4. DB (Sub, Inv) of FEV1% predicted within the period 5. Treatment from baseline to week 24; .Glie l iciiae ipyiaAt 82(02 690 (2012) 1822 Acta Biophysica et Biochimica / al. et Galli F. • Treatment changes with respect to the variables:spirometry, peak flow, quality-of-life, weight/height, percentage of neutrophils/other cell types (induced sputum), induced sputum levels of GSH/inflammatory mediators, pulmonary exacerbation 2 A phase I study of inhaled Billings United States Terminated Drug: A|S I 1. R|E • Assessment of safety of inhaled sodium Feb-06 00332215 sodium pyruvate for the M.C.E. • Inhaled sodium pyruvate 70 2. Safety/eff pyruvate in Subs with CF. Subs will be n.a. treatment of CF 3. PA evaluated for the presence of symptoms 4. DB and safety laboratory measurements. 5. Treatment • Determination of improvement in lungs of CF Subs as determined by measurement of FEV1 and measurement of inflammatory markers in induced sputum. 3 Inhaled GSH versus Marsico Italy Recruiting Drug: C|A III 1. R|E • FEV1% Jun-10 01450267 placebo in CF S. • Inhaled reduced GSH 150 2. Eff • Small airway function; exercise capacity; Dec-12 • Physiological solution 3. PA BMI; dyspnoea; cough; quality of life; 4. SB (Sub) pulmonary exacerbations; markers of –

5. Treatment oxidative stress (H2O2)in serum and in 713 EBC; epithelial inflammatory markers on BNEC 4 Safety and efficacy of Sagel United States Completed Dietary supplement: C|A II–II 1. NR|E • Plasma levels of β-carotene Aug-07 01018303 an antioxidant-rich S.D. • AquADEKs 17 2. Safety/eff • Plasma levels of coenzyme Q-10, Nov-09 multivitamin 3. SGA retinol (Vitamin A), 25-hydroxy vitamin supplement in CF 4. OL D, α- and γ-tocopherols (Vitamin E), 5. Treatment PIVKA-II 5Efficacy and safety of Kerem Israel Not yet Dietary supplement: A|S n.a. 2. Safety/eff • Changes in nasal chloride secretion as Sep-09 00889434 epigallocatechin gallate E. recruiting • EGCG 18 3. CA assessed by TEPD, with assessment of Jun-11 (EGCG)/tocotrienol in • Tocotrienol 4. OL mean changes in TEPD by drug compared 18 patients with splicing- • EGCG+tocotrienol 5. Treatment to baseline and the proportion of patients mutation-mediated CF with a chloride secretion response by drug compared to baseline • Pulmonary function testing: FEV1, FVC, MEF25-75 6 The effect of inhaled Van Belgium Terminated Drug: C|A IV 1. NR|E • Changes in visco-elasticity and Jan-10 00996424 NAC compared to Daele S. • Acetyl-Cys 19 2. Eff lung function Dec-10 normal saline on • Normal saline 3. CA sputum rheology and 4. OL lung function 5. Treatment 7 Safety of orally Goss C. United States Completed Drug: AI1. NR|E • Safety and tolerability of 14 days of Apr-05 00219882 administered • Standardized turmeric 11 2. Safety/eff treatment with orally administered Jan-06 curcuminoids in adult root extract 3. SGA curcuminoids as assessed by adverse subjects with CF 4. OL events, laboratory parameters, and 5. Treatment spirometry. • Pharmacokinetics of repeated doses of orally administered curcuminoids; change in NPD measurements. change in sweat chloride measurements. 8 Nasal potential studies Rowe S. United States Recruiting Other: quercetin C|A II 2. Eff • NPD(NPD) Biomarker Mar-10 01348204 utilizing CF transmembrane 46 3. SGA • Residual CFTR activity Nov-11 regulator (CFTR) modulators 4. OL 5. BS 9 Safety and tolerability of Sagel S. United States Completed Drug: C|A|S II–II 1. R|E • Safety and tolerability of drug, Jul-04 00570349 inhaled nitric oxide in • Nitric oxide for inhalation 18 2. Saf assessed by change in methemoglobin Dec-08 patients with CF • Nitrogen 3. PA levels, oxygen 4. DB (Sub, Care, Inv, OA) saturation, FEV1 5. Treatment • Assess the difference in sputum bacterial density before and after .Glie l iciiae ipyiaAt 82(02 690 (2012) 1822 Acta Biophysica et Biochimica / al. et Galli F. NO inhalation, and the difference in lower airway inflammatory measures before and after NO inhalation 10 NAC Phase IIB: a multi- Conrad United States Completed Drug: C|A|S II 1. R|E • Change in the logarithm of the level Nov-08 00809094 center, phase IIB, C. • NAC 80 2. Eff of human neutrophil elastase Feb-11 randomized, placebo- 3. SGA (HNE) activity measured in sputum controlled, DB study of 4. DB (Sub, Care, Inv, OA) • Change in concentration of IL-8 the effects of NAC on 5. Treatment measured in sputum and plasma; redox changes and lung concentration of GSH measured in inflammation in CF patients whole blood; the neutrophil count measured in sputum 11 Glutamine supplementation n.a. United States Not yet Dietary supplement: A|S II 1. R • Percent increase in plasma glutamine Feb-10 01051999 in CF recruiting • Glutamine 40 3. PA and GSH redox levels measured at Feb-11 • L-alanine 4. DB (Sub, Care, Inv, OA) weeks 0, 4, 8, and 12. 5. Prev 12 Effect of sulforaphane in Chmiel United States Active, not Dietary supplement: A n.a. 3. SGA • Nrf2 activation in NEC Apr-11 01315665 broccoli sprouts on J.F. recruiting • Broccoli sprouts 15 4. OL • Measures of lipid peroxidation in NEC; Sep-11 Nrf2 activation 5. BS GSH from blood lymphocytes; oxidative stress in urine; neutrophil migration into the –

gingival crevices 713

Abbreviations: A=adult; BC=breath condensate; BNEC=brushed nasal epithelial cells; BS=basic science; C=child; CA=crossover assignment ; Care=caregiver; CF=cystic fibrosis; DB=double blind; EBC=exhaled breath condensate; Eff=efficacy study; FEV1=forced expiratory volume in 1 s; FVC=forced vital capacity; GSH=glutathione; Inv=investigator; MEF25–75=maximal expiratory flow 25–75; n.a.=not available; NAC=N-acetylcysteine; NEC=nasal epithelial cells; NLF=nasal lavage fluid; NPD=nasal potential difference; NR|E=non-randomized|endpoint; OA=outcomes assessor; OL=open label; PA=parallel assignment; PI=principal investigator; Prev=prevention; R|E=randomized| endpoint; S=senior; Saf=safety study; SB=single blind; SGA=single group assignment; Sub=subject. a Search was done matching the terms “antioxidant therapy” or “Glutathione” with “Cystic Fibrosis”. b 1. Allocation; 2. Classification; 3. Intervention model; 4. Masking; 5. Primary purpose. 697 698 F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 corrected Cftr KO-transgenic mice were given an oral bolus dose of antioxidant transport, such as CF, might lack the ability to adapt to GSH (300 mg/kg) and compared with WT mice for changes in GSH the infection which may lead to a more severe inflammatory levels in plasma, lung, ELF, and BAL cells. There was a twofold in- response. crease in plasma, a twofold increase in lung, a fivefold increase in ELF, and a threefold increase in BAL cell GSH levels at 60 min in WT 3.2.2. Clinical trials on GSH mice; however, GSH levels only increased by 40% in the plasma, 60% The discovery of the defect in GSH export has suggested that ther- in the lung, 50% in the ELF, and twofold in the BAL cells within the apies able to restore or increase GSH levels in the ASL could counteract gut-corrected Cftr KO-Tg mice. No change in GSH levels was observed the inflammation and oxidative stress conditions typical of CF pa- in the uncorrected Cftr KO mice. These studies suggest that oral GSH tients. In an attempt to strengthen extracellular defenses against administration can increase plasma and lung compartment GSH ROS, some pilot studies have analyzed the effect of GSH inhalation or levels in WT mice and to a lesser extent in gut-corrected Cftr KO-Tg that of oral GSH prodrug N-acetylcysteine (NAC). All these treatments animals. It also suggests that oral GSH treatment can boost BAL cell were well tolerated by the CF patients and most authors were able to GSH levels. However, since this study failed to show significant in- measure increased ELF concentrations of GSH in association with creases in serum and lung compartment GSH levels in uncorrected some positive clinical outcomes [40,41,73,78,96,122–128]. Although Cftr KO mice, it is questionable whether oral GSH administration to potentially promising, these findings need stronger clinical evidence CF patients with intestinal malabsorption would benefit from this in that the majority of these were obtained on very limited number therapy. It was also shown that GSH is rapidly distributed to the of patients investigated in non-randomized controlled trials. This is serum and lung compartments. Kariya et al. [118] speculate that highlighted in a recently published meta-analysis [129] that provides other transporter(s) besides CFTR are responsible of the transport of also a thoroughly analysis of the literature on this aspect of the antiox- GSH and probably of other dietary molecules to the lung, which idant and anti-inflammatory therapy of CF. Among the American CF may be responsible for dietary deficiencies observed in various lung foundation sponsored trials two phase II trials on inhaled GSH and diseases. oral NAC are in progress in US and Germany (ClinicalTrials.gov, Iden- Another strategy is represented by the oral supplementation of tifier: NCT00506688 and NCT00809094, respectively; Table 1), and high doses of NAC, a well known cysteine donor for the synthesis of safety and tolerability of aerosolized glutathione is also matter of glutathione. NAC is considered a safe molecule, which has been investigation by another (not registered) US trial (more information used successfully to treat GSH deficiency in a wide range of diseases on this can be found in [130]). Nevertheless, the diffusion among CF [119]. As CF mice display defects in GSH export in the ELF comparable patients of NAC preparations for inhalation (Mucomyst®) has in- to those of patients, they could provide a useful tool to assess the ef- creased in recent years. fects of NAC administration on the GSH status. However, only limited γ-Glutamylcysteine ethyl ester (GCEE) is another potentially inter- studies exploring the effects of NAC on CF animal models have been esting GSH pro-drug, which has proved some efficacy in the ameliora- so far carried out. The effects of NAC have been tested on mucus accu- tion of oxidative stress e.g. in experimental myocardial infarction mulation, bacterial load, transit, and inflammation in the CF mouse [131] and central nervous system conditions (see e.g. [132]). However, small intestine showing that NAC may reduce intestinal mucus accu- GCEE has not been investigated in CF yet. mulation bacterial overgrowth in the gut [120]. Moreover, NAC has been reported to restore the accumulation 3.3. Limits and potential problems associated to thiol-based therapies of unwanted/misfolded proteins in aggregates that are associated with the CF airway phenotype as a cause of lung inflammation [121].The Although the above cited clinical trials may be considered promis- mechanism of this NAC-derived effect seems to involve the restoration ing attempts to improve the antioxidant levels in the ELF, the actual of beclin 1 expression and activity in the autophagy pathway of the capacity of these treatments to produce positive clinical effects must endoplasmic reticulum that was investigated in vivo using Scnn1b- be considered with caution. For example, indices of oxidative damage transgenic and Cftr(F508del) homozygous mice. The restoration of were found to be unaffected by aerosolized GSH treatment [122]. this pathway also produced a rescued trafficking of CFTR (F508del) to Some in vitro studies have suggested that the reaction of GSH with the cell surface of CF cells obtained from human CF nasal biopsies. PCN could produce hydrogen peroxide, with potential exacerbation Given the defective GSH metabolism of CF reviewed in the previ- of oxidative damage [101]. The exact mechanisms of PCN toxicity ous sections and in [122], and the reduced response to GSH therapy and the reaction of this toxin with GSH must be better understood in CF mice [93], some Authors have investigated the influence of bac- in order to evaluate the safety of GSH administration to patients col- terial infections on lung oxidative stress. The effects of P. aeruginosa onized by P. aeruginosa. infection on ELF and lung tissue antioxidants and the oxidation of Inhalation of GSH ensures its direct delivery in the airways, but DNA and lipids were investigated in mice challenged with bacterial since GSH can rapidly convert to its oxidized form GSSG, frequent cells [90]. CFTR-KO (B6.129P2-Cftrtm1Unc) and WT mice were chal- GSH inhalations are required to maintain a high GSH/GSSG. As a con- lenged intratracheally with a clinical isolate of mucoid P. aeruginosa sequence of this limit, four separate inhalations have been used in embedded in agar beads and on the third day of infection BALF and the study carried out by Bishop et al. [123]. Unless justified by clear lung tissue were obtained and analyzed for cytokines, antioxidants clinical improvements, these repeated treatments may represent a and enzyme activities [90]. P. aeruginosa lung infection increased burden for patients already undergoing complex therapies. Moreover, levels of inflammatory cytokines and neutrophils in the ELF. This cor- repeated inhalations of GSH increase the levels of GSSG in ELF responded with a marked increase in GSH and in urate levels in the [133,134]. In the absence of effective homeostatic mechanisms ensur- ELF of P. aeruginosa-infected WT mice. A twofold increase in urate ing the fast recycling of GSSG to GSH, this might produce unwanted levels was also observed among lung tissue antioxidants of P. effects and even the exacerbation of CF symptoms being GSSG re- aeruginosa-infected WT mice. There were no changes in markers of sponsible of the S-glutathiolation and functional inhibition of CFTR lung oxidative stress associated with the P. aeruginosa lung infection. [124]. In contrast to WT mice, the CFTR-KO mice lacked a significant in- In principle, the oral administration of GSH could be considered a crease in ELF GSH when challenged with P. aeruginosa, and this corre- safe strategy to prevent GSSG accumulation, but this strategy is likely lated with a decrease in the ratio of reduced to oxidized GSH in the not feasible due to the substantial inability of this water-soluble mol- ELF, a marker of oxidative stress. These data would suggest that the ecule to cross biological membranes. Moreover, a study carried out in lung adapts to infectious agents with elevated ELF GSH and urate. mice has established that GSH absorption in the gastrointestinal tract, Therefore, individuals with lung diseases associated with altered if any, is mediated by CFTR itself, thus excluding the possibility to F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 699 improve circulating levels of GSH in CF patients through the dietary considered to be linked with a cause–effect relationship with oxidative supplementation of the antioxidant [127]. The limited absorption of stress and is proposed to influence morbidity and mortality in condi- GSH could be overcome by GSH-esters, as in vitro studies have tions associated with chronic inflammation and severe oxidative stress shown that this form of GSH may be specifically useful to rescue mi- [143,144]. The human serum albumin (HAS) molecule contains only a tochondrial defects in cystic fibrosis models [80]. Safety of these GSH reactive thiol group, e.g. the Cys 34, the importance of which as an anti- derivatives in humans has been poorly investigated. oxidant defense system in blood and for the entire organism is well Two independent studies have shown that the treatment of CF pa- documented [145]. This is the second main thiol (and the main protein tients with high doses of NAC increases extracellular GSH in sputum thiol) in the circulation (approx. 2 mmol in the adult organism, assum- [78,125], but contrasting results have been reported concerning the ing 5 l of total blood volume, an Ht of 40% and [HAS] of 45 g/l of plasma), effects of NAC on the concentrations of blood GSH and on the levels being the RBC GSH the first thiol in blood (near to 5 mmol, assuming the of IL-8 and other markers of inflammation. NAC treatments may be same parameters of above and an average concentration of GSH in useful to modulate the GSH content in cells but it should be reminded packed RBC of 2.5 mM) [146,147]. The antioxidant role of HSA is not that an enhanced cysteine supply cannot lead to an increase of GSH only a consequence of the relative abundance of its Cys thiol. A specific above physiological levels due to the feedback inhibition mechanism capability of acting as a sacrificial target for a series of electrophils and of γ-glutamylcysteine synthetase, described in the Section 3.1.1. most biologically relevant ROS, i.e. hydrogen peroxide and peroxyni- trite has been demonstrated in a series of studies [145,148,149].Li- 3.3.1. Gamma-glutamyltransferase and GSH therapy gand binding activity can contribute to promote antioxidant effects So far none of the studies mentioned above has taken into account by the HSA molecule [145]. Transitions metals, particularly copper the fact that GSH is degraded by GGT enzyme activity. GGT concentra- and also iron in the case of iron-overload diseases, bind to HSA. In tions are known to increase several fold in ELF of CF patients, even if this way, these are less available to promote the Fenton chemistry the mechanisms for this effect were not determined [135], and this and hydroxyl radicals eventually released from this oxidative reaction phenomenon parallels the above described decrease of GSH levels in are mostly directed to the HAS protein sparing more important tar- ELF. Preliminary data suggest that a major source of increased ELF gets. A free radical-trapping activity of HSA has been also demonstrated GGT is represented by activated neutrophils accumulating in diseased and this was proposed to be directed toward both hydrophilic and fat- airways (Corti and Pompella, 2011, unpublished observation). Re- soluble species. This activity may result from the interaction with gardless of its origin, it is likely that increased GGT in ELF would de- other antioxidants such as α-tocopherol [150] and may influence the grade locally administered GSH to variable extents, which could antioxidant activity of food-derived phenolic antioxidants [151]. contribute to the so far inconclusive results of therapies based on However, Cys 34 is considered the main contributor to the antiox- aerosolized GSH. Besides its role in GSH catabolism, GGT has been idant function of HAS, which plays its role in the antioxidant homeo- shown to mediate protein S-thiolation [136], suggesting that GSH ad- stasis of blood thanks to a complex series of interactions with the ministration in the presence of active GGT enzyme might alter CFTR metabolism and antioxidant function of the pool of free thiols in plas- glutathiolation status and function in a potentially unfavorable way. ma and in the circulating RBC, with the latter playing a significant On the other hand, a potential role of GGT in favoring bronchial up- contribution to the extracellular pool of GSH [146] and to the dynam- take of antioxidant vitamin C has also been suggested [137]. ics of inter-organ GSH metabolism in cooperation with liver and other These controversial findings may add further issues to the ques- tissues [147]. Immuno-inflammatory cells and the lung tissue are tion of whether the therapy of lung oxidative stress by aerosolized among the main terminals of this metabolism. The RBC contain the GSH could be safe in all the CF patients regardless of specific strate- entire machinery to synthesize GSH, to restore its redox (by enzymat- gies that would ascertain the extent of lung inflammation. These ic reduction of the oxidized form), and to use this as cofactor of Se- strategies should include the assay of GGT levels in ELF. In principle, GPx and GSH-S-transferase enzymatic activities that are responsible once an adverse role of ELF GGT in GSH therapies will be confirmed, of the detoxification of hydroperoxides and alkylating agents that the association of GGT inhibitors in the GSH formulations for inhala- may form in or enter the RBC cytosol [147]. In this sense, the RBC rep- tion could represent a promising pharmacological strategy. resents a circulating reservoir of GSH that, in addition to maintaining the redox and respiratory function of Hb, participates to systemic pro- 3.4. Malnutrition as a possible cause of defective thiol-dependent tection of xenobiotics of endogenous and exogenous origin. In consid- antioxidant protection eration of these aspects, the combination of hypoalbuminemia and anemia may exponentially increase the risk of developing oxidative Successful nutritional interventions strongly impact on the clinical stress in CF patients as it is supposed to occur in other oxidative stress outcome of CF patients [138–140]. Malnutrition by pancreatic insuffi- conditions such as chronic kidney disease [55,143,152]. Additional ciency and other CF-related factors influence the susceptibility to de- clinical investigation should verify the hypothesis that hypoalbumi- velop recurrent infections and severe inflammatory lesions of the nemia together with a defective uptake and metabolism of sulfur- lung tissue. If the onset of a defective (sub-optimal) antioxidant sta- containing amino acids and CF-specific defects of the GSH metabo- tus may represent an underlying component in the clinical effects of lism, may represent a causal risk factor for an impaired antioxidant malnutrition remains a matter of investigation. Clinicians have to defense and systemic oxidative stress in CF patients. pay particular care to avoid these disturbances and successful proto- cols of nutritional intervention in CF have been developed, which 3.5. Fat-soluble antioxidants are essentially aimed to avoid the onset of protein-energy malnutri- tion (PEM) [141]. Despite this, the risk of developing such an unto- Pancreatic insufficiency and a diminished bile acid pool cause mal- ward complication in CF infants and children remains high as absorption of important essential nutrients and other dietary compo- suggested by the prevalence data registered in some areas. Actually, nents in CF. Of particular significance is the malabsorption of fat- a prevalence of PEM between 5 and 14% in Moldavian infants was re- soluble antioxidants such as tocopherols, carotenoids and coenzyme cently reported [142] and PEM is associated with poor outcome and Q-10 (Co-Q10), and that of essential fatty acids (EFA). particularly with the risk of developing edema and anemia. Besides lowered intake and absorption of micronutrient antioxi- 3.5.1. Vitamin E dants discussed in the other sections, malabsorption of dietary protein Vitamin E therapy in CF has been proposed in several decades of and excess fecal amino acid losses result in hypoproteinemia/hypoalbu- research as a useful approach to overcome both the lower absorption minemia as key biochemical signs of PEM. Hypoalbuminemia is of this fat-soluble micronutrient and the increased antioxidant 700 F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 demand by the abnormal generation of ROS in CF tissues (see for in- in CF. As further addressed below, this aspect could be the main limit to stance the recommendations by the Cystic Fibrosis Foundation Con- a successful use of natural forms of vitamin E in the clinical manage- sensus Conference on nutrition [153]). ment of CF inflammation even if a local lung-targeted therapy would The first report of a vitamin E deficiency in CF appeared in literature be developed according with so far proposed pre-clinical models of in 1951 by Filer et al. [154]. In this study, the absorption of the main aerosolized vitamin E [164,165]. form of this vitamin, e.g. α-tocopherol, and its ester derivatives was in- Current pharmacological research is aimed to develop synthetic forms vestigated in several subsets of infants and children. Thanks to a simple of this and other fat-soluble antioxidants with better radical scavenging bioavailability (or tolerance) test, these authors observed that “in- properties at the lipid–water interface. Type of ROS target and the sites fants… diagnosed as fibrocystic disease of the pancreas, diarrhea and of action greatly influence the chances of a fat-soluble antioxidant of alle- cirrhosis were characterized by a poor response to the test, i.e., the tol- viating oxidative stress in the airways as well as in other organs such as erance curve was low”. However, abnormalities were also observed in liver and pancreas. These aspects dealing with specificity of action other subgroups of infants with a variety of disorders not associated [163,166,167] have stimulated the search of novel vitamin E-derived an- with fat or fat-soluble vitamin intolerance, such as sprue, celiac syn- tioxidants that may help to scavenge radicals at the lipid–water interface drome and lupus erythematosus, which suggests the general observa- of the epithelial cell membrane and surfactant. Amine derivatives tion that malnutrition and inflammatory and degenerative diseases of of tocopherols and tocotrienols have been recently demonstrated to the GI tract, may lead to absorb tocopherols poorly. At the same time, show higher antioxidant and free radical scavenging activity than α- these authors reported that “Metabolic disorders with associated hy- tocopherol [168]. Further in vitro pharmacological analysis has included percholesterolemia were observed to give abnormally high values for toxicity evaluations and the detailed investigation of scavenging of azo- the area under the curve” and that patients responding poorly to to- and phenolic radicals with different degrees of hydrophobicity, and the copherol absorption test did also in vitamin A absorption tests. inhibitory activity on IL-8 gene expression and phospholipase activity The finding of lowered (lipid uncorrected) levels of vitamin E was in CF cells. Comparative evaluation with other synthetic derivatives, confirmed in other studies in which this relative deficiency was found such as α-tocopheryl succinate and natural forms of vitamin E, suggested to occur irrespective of pancreatic comorbidity and in association that these amine derivatives are promising antioxidant and anti- with lowered levels of other liposoluble vitamins such as vitamin A inflammatory agents [Galli F. and Pilolli F., unpublished observation] and D [155] regardless of their different liver metabolism and tissue deserving further pre-clinical investigation in CF model systems. delivery mechanisms [156,157]. Anti-inflammatory effects of natural and synthetic analogues of vi- Plasma, buccal mucosal cells (BMCs), and breath condensate α- tamin E are also an intriguing pharmacological opportunity currently tocopherol decreased significantly with age in association with a de- under investigation by several laboratories [169,170]. creased respiratory function [5]. This was accompanied by lowered Recently, vitamin E supplementation has been at the center of a levels of other antioxidants such as vitamin C, and increased oxidative dispute regarding its safety when used at high dosages in certain stress markers of different origin such as protein carbonyls, thiobarbi- populations of patients. An extensive and speculative debate originat- turic acid-reactive substances, and F2-IsoPs. ed on this subject after a meta-analysis study by Miller et al. [171] that Clinical symptoms of vitamin E deficiency in CF have not been examined the largest secondary prevention trials on vitamin E trials in conclusively investigated. Dolan et al. [158] described that anemia cardiovascular patients finding a significantly increased mortality risk of CF patients is related to vitamin E deficiency and increased for all the causes (about 4%, 1–8% in the 95% interval of confidence) peroxide-induced hemolysis of RBC. Other authors, however, ob- when the patients were treated with doses >400 IU/die (that are served an increased susceptibility to peroxide-induced hemolysis equivalent to 400 mg/die of the synthetic form allrac-α-tocopherol also in the presence of normal levels of vitamin E [159]. Peters and and to 185 mg/die of the natural form RRR-α-tocopherol). This debate Kelly [160] observed that RBC vitamin E concentrations were below resulted in a careful examination of this meta-analysis study by several the normal range in almost all unsupplemented patients and rose other authors and in further revisions of the literature on vitamin E tox- into the normal range with a 1-year supplement of 100 mg per day, icity in humans [see the literature recently reviewed in [163,172–174]] but not 15 mg per day. Since RBC vitamin E concentration has been that clearly demonstrated the poor consistency of the conclusions shown to correlate well with tissue concentrations of the vitamin in raised with Miller's meta-analysis study and the paucity of the concerns animals, tissue levels of vitamin E are expected to be lower than nor- that derived from that. These conclusions have been verified in the re- mal in CF patients. cent international symposium on vitamin E of the Society for Free Rad- Bioavailability of fat-soluble vitamin is limited in CF. Vitamin E ical Research, Europe branch of Rome 2009 [173,175].VitaminEusedas deficiency of CF is also associated with hypocholesterolemia [7].As supplement for humans in all its forms (e.g. α-tocopherol and other a consequence, the relative deficiency of this vitamin is compensated tocopherols and tocotrienols) is safe in abroad range of intakes when the absolute levels are normalized for cholesterol levels, and [172,176]. The tolerable upper intake level (UL) and the Acceptable this suggests a poor transferring of this vitamin in the circulation by Daily Intake (ADI), established by the Joint FAO/WHO Expert Committee a defective lipid and lipoprotein metabolism. Pancreatic insufficiency on Food Additives for the natural form of vitamin E as α-tocopherol and the consequent lipid malnutrition cannot completely explain this equivalents are of 300 mg/die and 0.15–2.0 mg/kg body weight/die, defect and other, possibly CF-specific, dysfunctions could play a role. respectively [172,177]. Liver metabolism and specific plasma transport systems of this vita- min need further elucidation in CF. 3.5.2. Carotenoids As a consequence of these aspects, it is not presently clear which Levels of plasma carotenoids such as β-carotene, β-cryptoxanthin, form and level of supplementation of this vitamin is most appropriate and total lycopene are significantly lowered in CF patients and this to treat these patients. Using α-tocopherol as a vitamin E supplement, was associated with higher susceptibility to lipid peroxidation Peters et al. [160] reported that 100 mg per day are required to nor- [5,178–180]. Rust et al. [178] demonstrated that the long-term oral malize RBC concentrations. Other authors described unsuccessful supplementation with 50 mg β-carotene/day (i.e. 1 mg β-carotene/ supplementation protocols with higher doses and this has led to de- kg BW/day) restored the levels of this carotenoid, while sub-optimal velop formulations with higher bioavailability in order to achieve bet- supplementation was observed at doses of 10 mg β-carotene/day or ter compliance to oral supplementation (see below and the literature lower, thus confirming the need of high doses of this fat-soluble factor reviewed in [161–163]). to overcome the limited absorption and thus to achieve plasma con- Besides absorption and tissue delivery issues, specificity of action centrations of healthy control subjects. Successful high-dose treat- is another critical item of antioxidant therapy with fat-soluble agents ments appear to lower oxidative stress markers such as TBA-MDA F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 701 complexes and to correct total antioxidant capacity of plasma. In dose of 2400 mg and the higher plasma concentrations were found another study, β-carotene supplementation was observed to de- to facilitate uptake by peripheral tissues and also the brain [186,187]. creased lipid peroxide formation as quantitated by malondialdehyde Laguna et al. [190] recently investigated total serum levels of concentrations in plasma (TBA/HPLC method), and to enhance the re- coenzyme Q-10 in a wide population of CF children (n=381) and esti- sistance to copper(II) ion-induced oxidation of low density lipopro- mated their association with clinical outcome. Near to 50% of these CF teins [180]. patients were deficient of Co-Q10 and this defect was significantly At the same time, toxicity issues have been raised for human sup- more prevalent in patients with pancreatic insufficiency and signifi- plementation with carotenoid formulations and particularly of water- cantly associated with P. aeruginosa colonization in infants (under miscible formulations of preformed vitamin A that is regularly sup- 24 months of age). Importantly, low Co-Q10 levels correlated to other plemented to CF patients, which may increase serum retinol and lipid markers of a poor nutritional status, such as total lipids and also possible risk of CF-associated liver and bone complications (reviewed the other fat-soluble antioxidants β-carotene and α-tocopherol, in [161,181]). However, β-carotene supplementation seems to be safe which confirms the presence of a common defect in the absorption since this does not affect plasma concentrations of other carotenoids and metabolic pathways of this coenzyme with dietary lipids. and retinol, as well as of other fat-soluble vitamins as α- and γ- The deficit of Co-Q10 may contribute to the impaired energy func- tocopherol [178]. Recent studies designed to test the clinical efficacy tion of mitochondria of CF tissues and this may exacerbate CF-linked of a CF tailored multivitamin formulation (commercial name AquA- inflammation, infection and cellular stress response of the lung. A sys- DEKs®), also tested the safety of this type of formulation and demon- tematic analysis of molecular lesions in CF bronchial tissue has been strated that this does not increase vitamin A above the normal levels recently carried out by proteomic approach [191]. Comparative eval- observed in healthy controls [162,182,183]. The normalization of β- uation of protein expressional pattern in CF and healthy control carotene levels obtained in these studies was associated only with tissues has revealed aberrant levels of some mitochondrial and minor improvements on respiratory and growth parameters, while energy-related proteins in CF specimens that included the ubiqui- the levels of urinary F2-IsoPs used as index of lipid peroxidation nol–cytochrome c reductase complex core protein I and one form of were not affected [162]. The surrogate marker of lipid peroxidation nidogen, a pseudogene of aconitase 2. These changes in CF may reflect MDA was affected together with some selected antioxidant parame- molecular changes which could be associated with an altered mito- ters (RBC thiols and superoxide dismutase) in another study in chondrial homeostasis and Co-Q10 redox. which this multivitamin formulation was preliminarily evaluated in Multivitamin supplements with high bioavailability containing comparison with standard formulations of vitamin E and A [182]. Co-Q10 have demonstrated to correct the deficit of this antioxidant and were preliminarily observed to improve airway inflammation markers in CF patients [183]. However, further clinical investigation 3.5.3. Coenzyme Q-10 failed to demonstrate that such an improved biochemical profile is Coenzyme Q-10 (Q10) is a well-known electron transporter in the associated with significant improvements in weight percentile and mitochondrial respiratory chain with fundamental role in cellular pulmonary function [162]. bioenergetics and scavenging of radical species [184]. This lipophilic substance is present in the circulation at low levels (serum concentra- 3.5.4. Fatty acids tions≤2 μM) mainly as ubiquinol-10, e.g. the reduced form, with an A key pathophysiological role in sustaining inflammation in CF has approximate ratio of 95:5 with the oxidized form ubiquinone-10 been attributed to the abnormal polyunsaturated fatty acid (FA) pat- [185,186]. A mechanism for a preferential distribution and accumula- tern. Abnormalities in FA profiling are potentially linked to CFTR tion in mitochondria has been suggested for both the reduced and ox- mutation-driven alterations in the absorption and/or metabolism of idized forms of CoQ10 that are taken up by the cells in a time- and dietary lipids [192,193], and to the consumption of high oxidizable concentration-dependent. Subcellular localization and trafficking of FA involved in the free radical-mediated lipid peroxidation [7]. exogenous Q10 are similar to those of the endogenous form, but Among those alterations, dysregulation of the docosahexaenoic acid were different from that of α-tocopherol that is related with lipid and arachidonic acid balance has been extensively studied with re- composition, particularly in the mitochondrial and microsomal frac- portedly significant reduction of DHA in CF and a parallel increase tions [184]. Ubiquinol-10 readily oxidizes ex vivo by the reaction in the levels of AA and inflammatory indices [193–195]. Actually, AA with other lipophilic antioxidants such as α-tocopherol and butylated is the progenitor of both enzymatic- and free radical-derived inflam- hydroxytoluene [185]. Therefore a higher reduction potential than matory mediators, including leukotrienes, prostaglandins and iso- other physiological fat-soluble antioxidants such as vitamin E and a prostanes. On the other hand, n-3 PUFA (eicosapentaenoic acid and selective metabolism and cellular trafficking, show peculiar role for docosahexaenoic acid) are involved in the generation of potent medi- the cell CoQ which may also represent an important lipophilic antiox- ators, namely resolvins and protectins, which are able to resolve idant in cells and body fluids. exudates and to act as organ protective and antifibrotic. Secondarily Human cells synthesize this coenzyme through the cholesterol bio- to their anti-inflammatory action, n-3 FA may also produce an synthesis pathway so that more than two thirds of the tissue levels ap- antioxidant-like response (e.g. a reduced demand of antioxidants to pear to have an endogenous origin; dietary sources provide a achieve an optimal control of oxidative pathways). As a consequence, contribution to Co-Q10 levels of blood and all solid tissues that varies n-3 PUFA have been suggested and widely used as supplements in CF depending on the dose applied and type of dietary source [187]. How- patients usually under the form of fish oil [196–198]. It is worth of ever, in the case of oral supplements, it has to be considered that hy- note, however, that defective levels of DHA in CF patients was not drophobicity and large molecular weight of this coenzyme influence confirmed in recent studies [7]. It was also shown that patients on its absorption that ultimately is slow and limited [186,187]. Likewise DHA supplements did not have increased plasma n-3 FA concentra- to vitamin E pharmacokinetics [188,189], Co-Q10 showed T(max) of tions, but showed more severe oxidative stress compared to the around 6 h that coincides with that of dietary lipids. Elimination is unsupplemented patients [7]. This observation of an increased risk close to that of the more retained form of vitamin E in human body, of oxidative stress in CF subjects receiving n-3 fatty acids supple- e.g. α-tocopherol, with a half-life of about 33 h, which suggests poor ments has also been described by other authors [199]. hepatic metabolism. However, commercially available formulations Studies reporting increased AA levels in CF patients have been are reported to be safe even at high doses and solubilized formulations contradicted by others [7,200,201] thus contributing to weakening show enhanced bioavailability. In healthy subjects plasma Q10 re- the pathophysiological role of the altered DHA–AA balance as turn sponse to oral ingestion show saturation profiles with a plateau at a point of an upregulated inflammatory status in CF. In this context, a 702 F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 recent Cochrane meta-analysis on n-3 supplementation in CF patients estimated rate of 5 μmol/l/yr and vitamin C concentrationsb40 μ- [202] highlighted the lack of evidence for a significant correction of mol/l were associated with highest indexes of inflammation which the assessed clinical end points (mainly respiratory symptoms) is consistent with the hypothesis that optimal levels of vitamin C even when inflammatory indices and other laboratory end points may influence immuno-inflammatory activity of alveolar macro- were met. Taken together, these data suggest that there is insufficient phages and neutrophils. Other few studies have examined the effect evidence to draw firm conclusions or recommend routine use of n-3 of supplements containing vitamin C on CF inflammation and oxida- supplements in CF. Notwithstanding, it is common belief that n-3 tive stress since the levels of this vitamin do not significantly improve supplements provide some benefits for people with CF with relatively with supplementation (reviewed in [213]). This highlights the com- few adverse effects and thus their use is not discouraged. mon fate that this hydrosoluble vitamin shares with several fat- Further alterations in fatty acid metabolism have been highlight- soluble counterparts in the “micronutrient paradox” of CF patients in ed, including the consistent findings of an increase in circulating which the need for a correction of their status along the progression levels of saturated and monounsaturated fatty acids [7,203,204]. of the disease is frustrated by the poor efficacy of oral supplementa- Decreased levels of essential FA (EFA), i.e. the FA that have to be in- tion protocols. Formulations and appropriate supplementation proto- troduced with the diet, correlates with the severity of respiratory cols that may produce a better correction of vitamin C status of CF insufficiency and the same clinical correlation was observed with al- patients are awaited for further clinical evaluation. tered proportions of FA species converted by the activity of desatur- ase enzymes (reviewed in [204]). The close relationship between 3.6.2. Selenium and selenium-dependent peroxidases certain fatty acids and oxidative stress, including the negative correla- Selenium is a trace element with marked electrophilicity [214] tion of C24:0 and linoleic acid with oxysterol levels point to the need that once converted to the organic form of Se-Cys can be introduced of intensive investigation in CF patients of previously neglected lipid in protein structures to play its important role in H2O2 metabolism species that are emerging candidates in the control of metabolism. and signaling [215]. As catalytic center of the enzyme GSH- Quantitative lipidomic analyses have lead to identify C16:1n7 palmi- peroxidase (SeGSH-Px), Se plays a crucial role to protect polyunsatu- toleate as a “protective” adipose-derived lipid hormone that strongly rated lipids of plasma membrane and circulating lipoproteins form stimulates insulin activity in muscle and liver, also suppressing in- peroxidative insults. With other Se-proteins such as the high molecu- flammatory cytokine output from mice fat cells [205]. Specific meta- lar weight thioredoxin reductases, this oligoelement participates to bolic activities have been also demonstrated by medium chain the control of protein thiol-disulfide oxidoreduction and glutathiony- saturated fatty acids, caprylic acid (C8:0), capric acid (C10:0) and lau- lation, which regulate signaling pathways of crucial importance in the ric acid (C12:0). Capric acid acts as a direct ligand of PPARγ, using a regulation of immunity and inflammation [16] but also the function- binding pocket different from the binding pocket of thiazolidinedione ing of other redox-sensitive proteins such as the same CFTR [124]. or long chain fatty acids [206]. Additional activities of medium chain The activity of SeGSH-Px in blood is considered a functional as- fatty acids, which are ligands of free fatty acids receptors detected sessment of selenium status, even if this assumption has some limita- in the immune cells, the gastrointestinal tract, and adipocytes, may tions related with the saturation profile that the Se stores show at contribute to metabolic homeostasis and inflammatory responses increasing doses of Se administration. Investigations of the selenium [207]. These data underscore the importance of a lipid-mediated “en- status in CF patients have produced conflicting findings, which may docrine network”, demonstrating how specific alteration of one or depend on differences in dietary intake, ethnicity and environmental few serum lipids would be per se sufficient to influence metabolic ho- factors in the diverse patient populations (reviewed in [216,217]). CF meostasis. Given the relevance of this emerging information and the children have been reported to have lowered blood selenium and RBC alteration of lipid metabolism and inflammatory status in CF, fatty SeGSH-Px activity [218,219], normal plasma selenium and lowered acid lipidomics need to be deeply investigated in CF. RBC SeGSH-Px [220] and even normal levels of both these two param- Again, these studies have obvious nutritional implications. Satu- eters [221]. The supplementation with selenium in combination with rated fatty acids with chain lengths higher than C18 are poorly other antioxidants was observed to increase the concentrations of absorbed partly because they form insoluble calcium salts [208]. blood selenium that, likewise β-carotene and fatty acid, were posi- Medium chain saturated fatty acids are well known for being highly tively correlated with improved lung function [6]. absorbed through the intestine, providing rapid delivery of energy Foucaud et al. [218], observed that a defective selenium status via oxidation of the more hydrophilic short chains, and have been was associated with lowered levels of other antioxidants that contrib- suggested to provide proper nourishment in patients with CF [209]. ute to the anti-peroxidative activity of this microelement, such as Recent studies have shown that consumption of a high-fat diet rich vitamin E (reviewed in [222]), and the severity of this deficiency in medium chain fatty acids, as opposed to long chain fatty acids, was lower in children with pancreatic enzyme replacement and vita- does not lead to ectopic fat accumulation in skeletal muscle and min E supplementation. liver of both rats and mice [210]. In light of the close association be- Treatments to substitute for exocrine pancreatic insufficiency by tween nutritional status, inflammation and life expectancy in CF pa- pancreatic enzymes from animal sources, such as porcine pancreas, tients, the manipulation of dietary lipids in these patients must be have been confirmed to be a source of Se [220]. These affects RBC further explored as a possible strategy to provide adequate nutrition SeGSH-Px activity and plasma selenium concentrations, which has and better management of oxidative stress. to be taken into account when selenium supplements are given to CF patients. 3.6. Hydrosoluble antioxidants, oligoelements and enzymatic antioxidants Selenium has been used to develop a series of organoselenium compounds that may open the way to new therapeutic opportunities 3.6.1. Vitamin C in CF (see Section 3.9.2). These include GPX mimetic drugs and phase Vitamin C status in CF patients has been poorly investigated. Early II enzyme inducers, which may provide higher antioxidant activity of studies suggested a defective vitamin C status that was refractory to ASL and cellular protection effects in the airways. oral supplementation [211]. Other and more recent studies showed normal or slightly decreased levels of vitamin C in CF patients as com- 3.6.3. Zinc and copper pared to healthy controls, but age- and disease-related decline of this Zinc and copper (Zn and Cu, respectively) are present in many water-soluble antioxidant was reported in these patients [5,212]. proteins so that a deficiency of these trace metals could have pleiotro- In the study of Winklhofer-Roob et al. [212] on mid-European CF pic effects in humans. As regards antioxidant systems, these two oli- patients vitamin C concentrations decreased with age with an goelements contribute an important role being cofactors of two F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 703 isoforms of the superoxide dismutase enzymes, e.g. the extracellular carotene (all-trans isomer), 90 μg Se (as selenomethionine), and form, e.g. the EC-SOD or Sod 3 and the Cu–Zn-SOD or Sod 1 that is 500 μg vitamin A (as retinyl palmitate) in oil. Plasma oligoelements, found in several tissues and cells [223]. The EC-SOD exerts its antiox- and particularly Zn, were in the normal range at baseline (as com- idant role also in the lining fluids of the airways [224]. pared with the data reported in [229,230]) and were not affected by The notion that CF patients have defective concentrations of blood this multivitamin supplement. The same was found for the activity Zn and Cu is controversial. As far as Cu status is concerned, few data of RBC SOD and for plasma 8-iso-PGF2α as surrogate biomarker of have been produced and are available in the literature suggesting lipid peroxidation. the notion that CF patients develop a moderate copper deficiency In a recent non-randomized small population (n=21) case–con- [225,226]. That was essentially demonstrated on the bases of a defec- trol study in CF children, Zn supplementation was proven to produce tive activity of Cu-related proteins such as plasma ceruloplasmin, positive clinical effects in Zn-deficient patients [196]. The supplemen- diamine oxidase, and RBC SOD. This defect seems to be refractory to tation with 5 mg/kg Zn sulfate/day (maximum 150 mg), significantly Cu and Zn supplementation (see below). decreased the number of infections and increased the forced expira- More advanced studies have been carried out in the case of Zn sta- tory volume in 1 s; energy intake and growth parameters also im- tus in CF. Low plasma zinc concentrations were reported in approxi- proved. These parameters were unaffected in untreated patients mately 30% of young infants with CF identified by newborn except that in the case of the pulmonary function that decreased sig- screening [227], and an impaired zinc homeostasis in CF patients nificantly. These clinical observations on Zn supplementation need to was described by Easley et al. [228]. This is characterized by poor con- be confirmed in prospective double-blind randomized control trial. servation of the endogenous pool with fecal loss and impaired frac- tional absorption of zinc, which are the consequences of pancreatic 3.7. Appropriateness and targeting of antioxidant therapies in CF insufficiency and persisting steatorrhea. These studies suggested that in the clinical management of CF patients these defects can be The choice of the appropriate antioxidant and dose to correct a cer- at least in part corrected by exocrine pancreatic enzyme replacement. tain biomarker and its associated biochemical lesion is another impor- A series of studies by Van Biervliet et al. [229,230] demonstrated tant point that appears to have disregarded in many clinical studies. in a Dutch population of CF infants and children that serum Zn varies For instance, unlike vitamin E, vitamin C supplementation does not in an age-dependent manner, but remained unchanged with respect alter F2-IsoPs levels in humans (reviewed in [74]). This appears to to healthy control levels. In CF patients no difference in serum Zn con- be true also in the antioxidant therapy of CF in which vitamin C centration between pancreatic-sufficient or pancreatic-insufficient (300 mg/day) was administered together with other antioxidants patients was observed and no correlation was found with the nutri- that included a dose of vitamin E of 200 mg/day, without any signifi- tional status or height z-score. Importantly, in these studies a signifi- cant effect on the surrogate biomarker of lipid peroxidation F2-IsoPs cant correlation of serum Zn was observed with the fat soluble [6]. When carotenoids are used as supplements to prevent the damag- vitamins A and E, thus confirming the relationship between the ab- ing action of ROS in the CF airways, it is noteworthy that these are not normalities of lipid and Zn metabolism in CF. This suggests that co- particularly good quenchers of peroxyl radicals relative to phenolics supplementation of Zn and fat-soluble vitamins should be advised and other antioxidants, but are exceptional in quenching singlet oxy- in the presence of pancreatic insufficiency and persisting steatorrhea. gen, at which most other phenolics and antioxidants are relatively Neve et al. [219] in a study aimed to assess plasma and erythrocyte ineffective. Singlet oxygen is not a radical and does not react via radi- zinc, copper and selenium in CF children, showed that mean plasma cal mechanisms, but reacts mostly by the addition to double bonds, zinc and copper levels were not different from those in age- forming endoperoxides that can be reduced to alkoxyl radicals that ini- matched controls which confirms the observations by Van Biervliet tiate radical chain reactions typical of the peroxidative damage of PUFA et al. described above. However, plasma zinc concentrations de- [38]. In this case the analysis of F2-IsoPs, lipid peroxide formation and creased in patients with moderate-to-severe growth retardation and transition metal-induced oxidizability of lipoproteins [162,180] are all with severe pulmonary disease, but very low zinc levels occur sporad- appropriate to evaluate the effect of carotenoids in preventing lipid ically. Erythrocyte zinc and copper levels were significantly higher peroxidation. than normal, while RBC selenium was lower than in age-matched As far as the dose is concerned, clinical pharmacology of vitamin E controls. These trace element concentrations in erythrocytes were as an antioxidant was recently investigated by means of the effect on discussed in relation to the activities of the Cu/Zn-SOD and the Se- F2-IsoPs production, and doses of α-tocopherol of 1600 IU/day or enzyme GPx. This observation suggests a compensatory upregulation greater were found to be required to statistically affect plasma F2- of the erythrocyte Cu/Zn-SOD by the exposure of erythroid precursors IsoPs levels in hypercholeterolemic subjects [176]. Several vitamin E to ROS and/or other CF-related stressors. supplementation studies in CF patients have been performed with Best et al. [225] also studied RBC SOD activity using this as a bio- doses of lower than 300 IU/day regardless of the lowered absorption logical sensor of Cu status in CF patients. A lowered activity of this by pancreatic and liver defects, and the regular supplementation enzyme was reported in CF together with that of the other Cu- with these doses does not appear to correct lipid oxidation markers dependent enzyme plasma diamine oxidase, while plasma ceruplas- in CF [7]. min showed normal activity. Degradation rates of copper proteins In the clinical practice and planning trials it has to be taken into are known to be accelerated in conditions of copper deficiency, consideration that, in spite of substantial evidence supporting a which could explain the finding at least in part. Anyway, when Cu higher antioxidant demand in CF, interventions with several antioxi- and Zn were supplemented to CF patients either separate or in dant formulations produce poor responsiveness particularly in the combination (6 weeks of 3 mg copper/d as copper-glycinate and case of fat-soluble antioxidants, which are poorly absorbed [6,162]. 30 mg zinc/d as zinc-glycinate), any of the copper enzyme activities Since many oxidants and antioxidants are present in tissues was affected. Therefore the moderate copper deficiency of CF patients and biological fluids and these have different chemical and physical appears to be refractory to the intervention by increased copper and/ characteristics, the possibility to produce a successful therapy with or zinc intake. a single antioxidant molecule is too far to be realistic. Moreover, an- Erythrocyte Cu/Zn-SOD and the plasma levels of Cu and Zn were tioxidants act by multiple mechanisms in a single system or by a also measured in the study of Wood et al. [6] in which Australian CF different single mechanism depending on the reaction system patients (age>5 years) were treated with a high-dose antioxidant responding in a different manner to different radical or oxidant multivitamin formulation containing 200 mg vitamin E (as RRR-α- sources. Because multiple reaction characteristics and mechanisms tocopherol), 300 mg vitamin C (as sodium ascorbate), 25 mg β- as well as different phase localizations are usually involved, no single 704 F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 therapeutic approach will effectively prevent damage by multiple Recent multivitaminic formulations have been designed to contain ROS sources. The defect of physiological antioxidants may also interfere also the minor forms of vitamin E and specifically γ-tocopherol [238], with antioxidant therapies that are based on one or few exogenous the expected superiority of which with respect to formulations con- antioxidants. taining only α-tocopherol needs to be verified. As discussed above Moreover, several antioxidant vitamins such as vitamin E and ca- in the section dedicated to vitamin E, further advancements in the rotenoids have multiple natural forms with different bioavailability, therapy of CF inflammation and oxidative stress could be based on metabolism and bioactivity [157,167,231]. As far as the case of vita- synthetic forms and natural metabolites of this vitamin that have min E supplementation concerns, α-tocopherol was used as unique been recently identified to possess higher antioxidant and also anti- vitamer in the large majority of supplementation trials so far per- inflammatory activity than α-tocopherol (reviewed in [163,168]). formed and is the form used in the nutritional management of CF Pre-clinical investigation of these and other fat-soluble agents is patients. Moreover, several supplements contain the synthetic (or ra- currently addressed to develop formulations and administration pro- cemic) form of this vitamin that has lower bioavailability than the tocols that may increase the therapeutic efficacy in the airways. In the natural form. Other forms present in nature and abundantly con- antioxidant therapy of lung dysfunction in CF, local administration tained in vegetables such as tocotrienols and the less methylated protocols may overcome the limits of oral and systemic administra- forms of tocopherol are not represented in many of the supplement tion protocols, increasing bioavailability and providing targeted formulations so far available in clinical centers, and the supplementa- approach to inflammation and oxidative stress. Aerosolization of vita- tion with α-tocopherol also limits their bioavailability. These forms min E and other fat-soluble micronutrients is possible and may allow show markedly higher susceptibility to hepatic metabolism and bili- direct administration in the airways to prevent toxicity of smoke ary excretion with respect to α-tocopherol, e.g. the most represented which is closely associated with inflammation and oxidative stress form in blood and solid tissues, and if co-supplemented, these are [164,165]. To our knowledge, this as well as other strategies of local easily displaced during liver uptake and excreted with bile by administration such as instillation of solutions such as surfactant- means of competition with the same α-tocopherol. Notwithstanding, like solutions enriched of vitamin E, have not been previously inves- these minor forms have been proposed to play important physiolog- tigated in CF patients and other CF model systems. ical roles showing molecular characteristics, transcriptional effects On the contrary, inhalation is an administration route widely and antioxidant activities that clearly differentiate them in subfam- adopted in the case of GSH and NAC therapy described above. In ilies with distinct biological functions. Some of these, such as α- this context, Cys formulations alternative to NAC have been proposed tocopherol, e.g. the second vitamin E form as relative abundance in for use as antioxidant and anti-inflammatory agent for inhalation. blood, and its carboxyethyl-hydroxychroman metabolite [189], ap- Nacystelyn is a lysine adduct of NAC that thanks to a higher proton- pear to have health-related anti-inflammatory effects which are par- ation equilibrium shows increased water solubility and thus better ticularly relevant in lung protection [232,233]. This has suggested bioavailability. Nacystelyn has been described to influence IL-8 gener- that such a group of “non-α-tocopherol” forms may represent anoth- ation and the inflammatory signaling of bronchial epithelial cells er family of vitamins within the family of vitamin E with an impor- [239] and preliminary clinical evaluation has demonstrated the safety tant, but often missed, contribution to the proposed health effects of of this drug [240]. Further clinical trials aimed to assess the effect on this vitamin [163]. lung symptomatology of CF patients are needed. Direct administra- These aspects may thus limit the possibility to provide CF patients tion in the airways could also be adopted for micronutrient vitamins of optimal levels of this vitamin in all its components, even if they are with poor absorption and bioavailability due to GI defects. regularly treated with a vitamin E supplement, e.g. with α-tocopherol. Targeted antioxidant therapy with formulations with higher bio- availability and bioactivity has been anticipated in CF. New antioxi- 3.8. Clinical impact of antioxidant therapy in CF dant formulations have been proposed to overcome main limits of antioxidant therapies so far proposed for these patients. Water- Huge in vitro and pre-clinical evidence has provided the rationale miscible α-tocopheryl acetate containing polysorbate, propylene gly- to support clinical investigation of antioxidant strategies in CF. These col or polyethylene glycol as emulsifiers form micellar structures should aim to restore the oxidant–antioxidant balance of CF airway were suggested to providing greater bioavailability than the fat- challenged by chronic infection and inflammatory cell activation. soluble counterparts. These were commercialized with the brand Some observational trials have confirmed that antioxidants used names of E-vimin®, Cremophor® EL and Aquasol® E, but despite as either supplements to the diet or drugs for lung administration the original positive expectation [234,235], some studies failed to ob- by aerosolized formulations, may help in relieving progressive lung serve a higher response in vitamin E levels when these were com- damage and other adverse clinical events of CF such as poor growth. pared with fat-soluble formulations [236,237]. So far, few studies have examined with sufficient methodological In a pilot study by Papas et al. [183] a micellar formulation of fat- rigor the clinical efficacy of antioxidant therapy in CF. This was also soluble nutrients and antioxidants was found to improve plasma concluded in a previous review of the literature by Cantin et al. [12] levels of β-carotene, γ-tocopherol and CoQ(10), reducing at the that was published in the beginning of 2006 and is confirmed also same time some inflammatory markers in induced sputum, e.g. mye- in a recent analysis of clinical trials on antioxidant therapy in CF pa- loperoxidase and to a lower extent PMN elastase and total cell counts, tients [213] that examined the literature until September 2010 while lung function and sputum bacterial counts were unaffected. using as sources the databases of the Cochrane CF and Genetic Disor- The same group recently confirmed the possibility to increase the ab- ders Group CF Trials Register, PubMed, CINAHL and AMED. Useful in- sorption of fat-soluble micronutrients using formulations with higher formation was retrieved from just four randomized controlled trials bioavailability specifically designed for malabsorbing patients such as and one quasi-randomized controlled trial on vitamin C, vitamin E, CF patients that include also vitamin K and commercialized with the β-carotene and selenium used as supplements administered alone brand name of AquADEKs® [162]. Despite improved vitamin and mi- or in combination. Post-hoc data analysis that was possible only in cronutrient levels, in this non-randomized, open-label study, AquA- three studies on a total of 87 CF patients, showed the absence of DEKs® produced only modest improvements in weight percentile any significant improvement in lung function that was selected as pri- and pulmonary function. Another recent pilot observation [182] has mary outcome together with quality of life that improved in one trial. suggested the beneficial effect of this formulation on antioxidant and Secondary outcomes concerning laboratory indices of oxidative stress oxidative stress parameters of this oral supplement that surely deserves and antioxidant status showed several improvements. These included more clinical investigation by larger randomized controlled trials. an increase of RBC SeGPx by selenium supplementation done as F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 705 individual or combined supplementation, and increased levels of all and anti-inflammatory drugs. Between the multitudes of natural sub- plasma antioxidants, except vitamin C, that were observed in all the stances, most investigated ones include for instance tyrosol, hydroxy- trials. tyrosol, gallic, and caffeic acids, belonging to the family of phenols, The message from these studies to pediatricians and nutritionists while resveratrol of the stilbenes family, epicatechin, kaempferol, within the clinical staff of CF centers is that in the absence of solid ev- and cianidin are examples of flavonoids; examples of fat-soluble phe- idence regarding the clinical effectiveness of oral formulations of an- nolics are those of the family of tocols (vitamin E) and carotenoids. All tioxidant supplements in CF (including PUFA), their use as a routine these compounds show different metabolisms and signaling effects clinical practice should be considered with caution keeping clear in [246]. Antioxidant and cell protection mechanisms are associated mind that this should represent just a complement of one of the with different and sometimes concurrent processes as H-atom trans- most critical interventions in the life-long clinical management pro- fer and electron donation, radical trapping and chelation of transition gram of CF patients, e.g. providing an adequate nutriture [140].In metals that can be easily investigated at the chemical level [247]. Be- this context, compensating energy and protein malnutrition has to sides these direct antioxidant effects, metabolic responses and signal- remain the first goal of nutritional management programs. Providing ing effects of natural compounds are also important to produce higher intakes of antioxidant micronutrients with food naturally- second-generation responses that modify the level of antioxidant enriched (or functionalized) of micronutrient antioxidants should protection and the potential for ROS detoxification in cells and body be preferred in order to have the higher combination of positive nu- fluids. These derive from the transcriptional regulation of antioxidant tritional factors that are lost during refinement procedures and over- and pro-oxidant genes that coexist with the modulation of cytokine all industrial processing of dietary supplements. secretion and inflammatory enzyme activities. Measures of good practice in clinical management of micro- Some of these natural plant-derived antioxidants may provide fur- malnutrition in CF patients would include the support of specialized ther pharmacological activities of possible relevance in CF. This is the personnel and laboratories. The centers should have the possibility case, for instance, of the plant flavonoids genistein, apigenin, kaemp- to ascertain with appropriate nutritional and laboratory tests what ferol, quercetin and hesperidin [248–251] that fall in the pharmaco- the level of deficiency is for the different micronutrients that each pa- logical category of “potentiators” [252] in that these activate at least tient may suffer from during a dietary program. Monitoring dietary to a certain extent CTFR-mediated Cl currents in normal and CF airway intakes and blood levels of micronutrients is not sufficient to set a epithelium investigated either in vitro or in vivo in humans and ani- personalized nutritional intervention with supplements or functional mal models. Actually, as prototypal flavonoid-derived potentiator, foods. Sub-optimal intakes and the compliance to nutritional inter- the tyrosine kinase inhibitor genistein potentiates the gating of wild- ventions should be verified by means of selected biochemical and type as well as ΔF508 CFTR and G551D CFTR [253], but at higher clinical criteria. Unluckily, the latter are still not well established for doses it inhibits CFTR gating, which make this an unlikely clinical can- some micronutrients such as the fat-soluble vitamins E (reviewed in didate. Similar potentiation effects have been reported for the antiox- [163,173,241]) and K [242,243], and also for trace elements such as idant curcumin [250] that shows an additive effect with respect to Se [244]. For many of the antioxidants used as supplements in genistein-induced potentiation. Besides CFTR potentiation, which humans we still do not know exactly several of the biological effects does not appear to have a direct influence on GSH and nutrient trans- that these may provide when introduced in a healthy body and we port in the lung tissue, all these natural substances have been used know less what these can do in disease. These aspects hardly impact alone or in combination with other agents as in vivo enhancers of on the concept of optimal levels of intake that should be identified for lung antioxidant protection [246,254,255]. In the case of quercetin, each micronutrient on an individual basis. According with the aspects thiol-reactive phenolic [256], bronchodilation and anti-inflammatory discussed in the previous sections, optimal levels of intake for antiox- effects by a restoration of the Th1/Th2 balance have been reported idant micronutrients are different in CF patients and healthy subjects in inflammatory syndromes of the lung [255,257–259], but these and vary with age and several individual factors. effects have not been explored in CF. Pyocyanin toxicity (see above Lung administration of antioxidants by means of aerosolized for- in the Section 2) could represent an issue for the use of quercetin mulations, so far used for GSH therapy (see above), is a promising and other catechols in infectious diseases of the lung [260]. field of clinical investigation in CF. Toxicity issues are currently the Other natural antioxidants of potential interest in CF therapy in- main concern and the subject of clinical evaluation by several groups clude terpenoids [261] such as terpinen-4-ol, a major constituent (described above). of tea tree oil that is being investigated in the therapy of anti- Information on future directions of antioxidant therapy in CF can biotic resistant organisms, specifically against methicillin-resistant be inferred by the analysis of pending or recently completed clinical Staphylococcus aureus (MRSA) [262], a problem in CF [263]. Other trials that have been searched (December 2011) at ClinicalTrials.gov than being lipophilic antioxidants, terpenes can prevent oxidative database matching the terms “antioxidant therapy” or “glutathione” stress of the CF lung secondarily to the antibiotic effect thereby pro- with “cystic fibrosis”. Fifteen trials were retrieved and those directly moting a better control of immuno-inflammatory pathways and lung related to antioxidant therapy (n=12) are presented in Table 1 and antioxidants. include efficacy and safety studies of inhaled drugs as GSH, sodium pyruvate, NAC, and NO, and the dietary supplements: AquADEKs®, 3.9.1. Targeting NF-κB with natural compounds: resveratrol and plant an antioxidant-rich multivitamin supplement, epigallocatechin gal- extracts late (EGCG)/tocotrienol, curcuminoids, the amino acid glutamine, Natural compounds were long known to be useful in designing sulforaphane in broccoli sprouts. One trial is investigating the flavo- anti-inflammatory drugs and some of these could find an application noid quercetin as CFTR modulator. in the control of the transcription factor kappa-B (NF-κB) that may lead to develop advanced pharmacological tools to control inflamma- 3.9. Emerging antioxidant and anti-inflammatory approaches in CF tion and to produce higher antioxidant protection to CF airways. Inhibitors targeting NF-κB, including transcription factors decoy Mediterranean foods such as olive oil and red wine or several molecules (TFD), are potent down-regulators of gene expression plant extracts such as herbal tea or grape seed extracts contain a and release of IL-8 in CF cells infected with P. aeruginosa [264,265]. series of natural compounds with important antioxidant and NO Resveratrol (3,5,4′-trihydroxystilbene, “E” form) is one of the most scavenging activity that have been widely investigated for the pre- investigated natural antioxidant with proposed activity as NF-κB vention of oxidative stress-related diseases [38,245]. These have inhibitor. This is a phytoalexin present in large quantity in red wine, been long investigated as template molecules to develop antioxidant preferentially in the skin of grapes, being its concentration 706 F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713

50–100 μg/mg of fresh skin and 1.3–3 mg/l of red wine [266]. produce concomitant antimicrobial and antioxidant effects. This sys- Furthermore, resveratrol is a constituent of ‘Darakchasava’ (1.3–6mg/ tem described above in Section 2.2 represents an important compo- l), an ayurvedic medicine from India [267]. The antioxidant activity of nent of innate immunity with a role in bactericidal defense and resveratrol has been reported in several papers and occurs also in lung regulation of H2O2 metabolism of airways [32]. CF patients have also tissues, suggesting that resveratrol has potential as a therapeutic reported to show lowered LF levels in the ASL [37] that together agent in respiratory disease [268–270]. Resveratrol can be considered with a CFTR-dependent defective efflux of SCN¯ and LPO [36] may cre- one of the most interesting molecules from the natural world ate the environment for a sustained flux of H2O2 and for the genera- exhibiting strong antioxidant activity that at the same time is able to tion of highly reactive Fenton species [11,38]. An ideal therapeutic exert anti inflammatory activity through alteration of NF-κBfunctions intervention should restore the LPO-dependent generation of bacteri- and efficient inhibition of pro-inflammatory genes [271,272]. As far as cidal OSCN¯ from DUOX-derived H2O2 and SCN¯ in the ASL to control the effects on CF model systems are concerned, Cabrini et al. [26] at the same time the microbial biofilm and the flux of H2O2 into the reported the effects of resveratrol on P. aeruginosa infected CF IB3-1 ASL. An approach close to this therapeutic mechanism was recently cells. The results obtained demonstrate that resveratrol exhibits minor proposed for the drug Meveol®. This is a complex of LF and OSCN¯, effects of IB3-1 cell growth and important inhibitory effects on accumu- with proposed in vivo antimicrobial activity [285] to be evaluated lation of IL-8 and GRO-α mRNA, while minor effects on IL-6 gene ex- in clinical trials for the aerosol treatment of lung infections in CF pression were observed. These data support recently published patients. The role for this drug in the control of H2O2 levels of ASL observations suggesting inhibitory effects of resveratrol in infection- remains to be investigated. mediated inflammatory processes [273]. Interestingly, resveratrol has been reported as a potent inhibitor of NF-κB activity and a promising 3.9.2.2. Organoselenium compounds. Organoselenium compounds anti-inflammatory agent [272,274] also in combination with other mol- have been proposed as therapeutic agents useful in the antioxidant ecules [274,275]. However, in vivo translation of this pre-clinical on res- therapy of inflammatory conditions. These include GPX mimetic mol- veratrol is hard to be foreseen. This natural compound displays poor ecules such as Ebselen® (2-phenyl-1,2-benzisoselenazol-3(2H)-one) bioavailability [276] and further characterization of resveratrol pharma- or other and more recently developed molecules that are still in cokinetics is troublesome since it is hardly measurable in vivo [277]. pre-clinical steps of evaluation [286]. Selectively and specificity of ac- Furthermore, the regulation of inflammatory responses by resveratrol tion are main issues in the therapeutic mechanism of this candidate has been suggested to be far more complex than simple direct suppres- antioxidant drug that thanks to the electrophilic properties of Se sion of NF-κB activity. Actually some aspects of the immune response [214] reacts with thiols to reduce H2O2 and other species. Moreover, could be enhanced by this natural substance, which may lead to Ebselen® has been described to act as potent non-competitive inhib- hypothesize broad negative feedback events on immuno-inflammatory itor of extracellular nucleoside diphosphokinase (NDPK) having neg- pathways [278]. In any case, and with these cautions in mind, the pub- ligible effect on ecto-ATPase and adenyl kinase activities, which are lished observations on cellular model systems support the concept that other players of the metabolism of extracellular nucleotides [287]. resveratrol and resveratrol-containing formulations deserve further con- This enzyme together with other nucleoside di- and triphosphates sideration to determine their possible therapeutic values in CF. contributes to regulate several components of the mucociliary clear- Other inhibitors can be identified from systematic studies of natural ance process (MCC) that protects the lung against infections via acti- products and their ability to interfere with NF-κBactivity[279–281]. vation of epithelial purinergic receptors. The inhibition of NDPK by Recent studies have demonstrated that the whole extract of Aegle this GPX-mimetic drug may also impact on the energy status of endo- marmelos (Rutaceae) has strong inhibitory effect on the P. aeruginosa- thelial cells [287]. This aspect could be further investigated as a cellu- dependent IL-8 induction in human CF-derived bronchial IB3-1 cells lar protection mechanism in the pulmonary epithelium of CF patients, without affecting cell proliferation [280]. Molecular analysis of compo- being the control of cell energy of fundamental importance to prevent nents contained in A. marmelos extracts revealed that three major com- the activation of death pathways [288]. pounds, namely 5,6-dimethoxy-1-indanone, 2-hydroxy-cinnamic acid Further therapeutic mechanisms for these Se-derived drugs may and 5-methoxy psoralen (5-MOP), reproduce the inhibitory effect ob- derive from the marked activity as inducers of phase II enzymes served with the whole extract [280]. A further example has been recent- such as quinone reductase (QR) and glutathione-S-transferase (GST) ly published using extracts from bergamot (Citrus bergamia Risso). In [289]. This induction response also observed with other electrophils this study the extracts were characterized and the main detected con- and redox-active molecules, such as natural phenolic antioxidants, stituents were assayed for their biological activity [281]. The results could produce higher cellular protection by the acquisition of an obtained demonstrated that the extracts from bergamot epicarps con- increased capability to detoxify endobiotics generated during oxida- tain components displaying an inhibitory activity on IL-8. Particularly, tive stress and inflammation [246]. the most active molecules were bergapten and citropten. These effects have been confirmed by analyzing mRNA levels and protein release in 3.9.2.3. Melatonin and GSNO as NOx regulators. Increased markers of the CF cellular models IB3-1 and CuFi-1 induced with TNF-α or exposed NO-derived biological damage are present in the CF lung suggesting to P. aeruginosa. the abnormal reactivity of NOx in the inflamed lung of CF patients A parallel strategy linking NF-κB inhibition and down-regulation [52]. However, an impaired metabolism and biological function of of NF-κB-dependent functions is described in recently published NO in CF airways (see Section 2.3) suggests a cautious approach to studies based on a structured-based virtual screening (VS) of differ- the use of agents that may influence the levels and chemical behavior ently substituted furocoumarins and analogues against NF-κB, with of NO-derived species (NOx) with possible impact on the physiologi- the objective of selecting molecules able to inhibit the binding of cal roles of NO in vasodilatation, innate immunity and H2O2 metabo- this transcription factor to DNA [282,283]. Novel compounds were lism of the respiratory tract [290,291]. NO donors also appear to identified with this strategy as potent inhibitors of NF-κB dependent influence CFTR function of alveolar epithelium and gland serous biological functions, to be proposed for the control lung inflammation cells [292,293]. occurring in patients affected by CF [282–284]. Main physiological role of the pineal gland hormone melatonin is the synchronization of circadian rhythms, including the sleep–wake

3.9.2. Scavengers and regulators of H2O2 and NO cycle, but several other functions dealing with immunomodulatory, antioxidant and cellular protection effects have been identified 3.9.2.1. Lactoferrin and OSCN¯. The impaired DUOX/LPO system of CF [294]. Melatonin receptor stimulation may also influence cyclic AMP patients could be the target of therapeutic strategies that may signaling and the regulation of CFTR ion channel [295,296]. F. Galli et al. / Biochimica et Biophysica Acta 1822 (2012) 690–713 707

A recent randomized double-blind placebo-controlled study has laboratory biomarkers could provide critical information to establish examined the effects of short term melatonin administration (3 mg intervention criteria for an early nutritional management of newly di- for 3 weeks) on sleep and oxidative stress markers in CF [297]. agnosed patients [5]. It is expected that a timely secondary prevention According with the expected pharmacological effect of this hormonal strategy could influence the progression of CF symptoms thus substance, the treatment was successful in improving sleep indices. affecting with a self-feeding mechanism the same extent of malnutri- At the same time, nitrite levels determined in exhaled breath conden- tion and oxidative stress along the life of CF patients. Since several sate (EBC) were reduced to suggest a better control of NO metabolism factors contribute to impair the nutritional status of CF patients of CF airways (discussed above in Section 2.3), but these effects, how- (such as pancreatic insufficiency, chronic malabsorption, recurrent ever, were not associated with a significant correction of isoprostane sinopulmonary infections and progressive lung disease, chronic in- as lipid peroxidation marker measured in EBC. Increasing dosages of flammation, increased energy expenditure, suboptimal intake, multi- melatonin could be investigated for the pharmacological treatment therapy) careful monitoring of the antioxidant status should be of oxidative stress and immune dysfunction of CF patients due to recommended. According with international guidelines, fat-soluble the absence of adverse effects of this hormonal substance in humans. vitamin supplementation is of utmost importance in daily practice to- The S-nitrosation reaction of thiols is another interesting aspect of gether with energy intake requirements and pancreatic enzyme- NO pharmacology [53] that may find an application in the therapy of replacement therapy. Among these, vitamin E, β-carotene and ω-3 lung complications of CF. This has been the case, for instance, of the FA have observed to alleviate selected biochemical signs of oxidative S-nitrosothiol GSNO that has been used safely in human trials stress as measured for instance with well established laboratory indi- [298,299]. This endogenous nitrosothiol with proposed regulatory ef- ces of lipid peroxidation, and in some studies these effects were fect on the CFTR of lung epithelia [292,293], is well tolerated in pa- preliminarily associated with positive clinical outcomes. Notwith- tients with CF when administered by aerosolization and may standing, randomized–controlled clinical trials on antioxidant supple- produce better oxygenation [299] thereby suggesting that therapy ments (including ω-3 FA) so far carried out in CF have failed to aimed at restoring endogenous GSNO levels in the CF airway may conclusively demonstrate significant beneficial effects on respiratory merit further clinical evaluation. Since the administered GSNO de- symptoms and on the consequent impact that these have on the qual- composes as shown by the increased levels of expired NO in CF pa- ity of life of these patients (reviewed in [202,213]), which may provide tients under treatment with this nitrosothiol [299], further no support for the use of these supplements in CF. An absence of effi- investigation should be addressed to ascertain whether GSNO thera- cacy in prevention studies on antioxidant supplements has also been py may merely correspond to a GSH therapy. observed in other, and possibly less severe, oxidative stress-related NO dependent effects on CFTR function should be considered to conditions, particularly in the secondary prevention of cardiovascular evaluate mechanism of action and specificity of antioxidant therapies disease and some forms of cancer by vitamin E and other antioxidants that target NO metabolism and function in the CF airways. such as vitamin C, selenium and β-carotene (reviewed in [163]). Dif- ferent biases, however, have been identified in randomize large clini- 4. Conclusions and future perspectives cal trials that may account for these disappointing findings. These include patient selection criteria (early interventions and primary The presence of clinical and biochemical symptoms of inflammation prevention could have more chances of success than secondary and oxidative stress implies that CF patients have a higher demand of prevention), duration and doses of the treatment, use of wrong anti- antioxidant protection. The combination of the dysfunctional CFTR oxidant formulations and administration protocols, absence of verifi- with a lowered intake and absorption of dietary antioxidants produce cations of the biological compliance to the treatment, etc. The same sub-optimal levels of protection from both enzymatic and non- biases apply for the few trials carried out in CF patients. Thus, the ap- enzymatic defense systems. The former could be associated with a de- propriateness and efficacy of nutritional interventions with natural fective glutathione metabolism and lowered intake of sulfur-containing food-derived or synthetic antioxidants should be verified with respect amino acids and oligoelements such as zinc and possibly selenium, to the biological pathways of oxidative stress and clinical variables while fat-soluble antioxidant vitamins, such as vitamin E and caroten- that are identified as end-points. Profiles of blood and tissue antioxi- oids, and the water-soluble vitamin C, are lowered than in healthy sub- dants, as well as of reliable surrogate markers of oxidative stress, jects due to malabsorption and possibly higher consumption. have to be selected and determined in highly specialized laboratories The increased antioxidant demand in CF patients should be even with a well-established experience on these analyses. higher in the presence of complications of the lung, pancreas and Well-timed (early) interventions with appropriate antioxidant liver. Lung comorbidity is particularly important in this context formulations/protocols need to be proposed for the next generation being associated with recurrent infections, which results in the alter- of trials, and the development of novel CF-tailored antioxidant and nation of inflammatory exacerbates and chronic inflammation. These anti-inflammatory agents should be promoted. sustain the flux of ROS in the airways and promote the formation of As a consequence of these considerations, more clinical investiga- second-generation byproducts by the damage of biomolecules such tion is awaited to identify future successful approaches to the antiox- as PUFA and proteins, which may diffuse also at the systemic level to idant therapy as a measure to further enhance quality of life and the further promote adverse biological responses and toxic reactions. Pan- overall clinical outcome of CF patients. creatic insufficiency causes malnutrition and may lead to develop en- docrine and metabolic defects associated with diabetes of CF. These Acknowledgments events can be further aggravated by the concomitance of liver dys- function and a poor nutritional status. Malnutrition is strongly associ- We are indebted with the Italian Cystic Fibrosis Research Founda- ated with poor prognosis as assessed by pulmonary function and tion for the support that it provided with its grant program to the re- survival data [138–140]. The more aggressive nutritional interven- search activity of the authors of this paper. This is a scientific initiative tions seem to produce better clinical outcomes in these patients and of the “Working Group on Inflammation in Cystic Fibrosis” (Bragonzi the possibility of achieving higher antioxidant protection in well- A, Bravo E, Cabrini G, Conese M, Cuzzocrea S, Dechecchi MC, De Ales- nourished patients by dietary factors could be a key aspect to explain sandri A, Evangelista V, Galli F, Gambari R, Lambiase A, Lucidi V, such a clinical advantage. 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